Difference between revisions of "Team:UChicago/Notebook"

Revision as of 07:22, 18 September 2015

UChicago Summer 2015 Lab Journal

Courage is not the absence of fear, but rather the judgment that something is more important than fear. The brave may not live forever, but the cautious do not live at all.

-- Eduard Christoff Philippe Gerard Renaldi, Prince of Genovia, The Princess Diaries

Week 1:

Goals- take inventory, design constructs and primers, test competent cells

6/15/15

Worked on primer design quiz questions. Discussed shift of project direction with Justin based on recently published paper (Chen, 2015): http://advances.sciencemag.org/content/1/5/e1500358.full

Took inventory, created excel sheet online under Protocols folder “Genehackers 2015 Inventory”

Downloaded SnapGene Viewer to work on plasmid constructs.

6/16/15

New idea for output system -create one construct with SasA/RpaA as activator of output molecule, another with LabA/RpaA as inhibitor (negative feedback loop) of output molecule. Based on article (Taniguchi, 2010). Started design on constructs, possibly 5 in total:

1. Read-out activator (KaiCEE-RFP, RpaA, SasA)

2. Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)

3. Test gene (kaibc promoter, GFP)

4. Fusion SasA

5. Fusion KaiC-P

Last two based heavily on paper (Chen, 2015)

Met with Jennifer Moran -need to do safety training before autoclaving liquids and other procedures, will accomplish once more people are back.

To Discuss: Is it worth having negative feedback regulator of RpaA/ output molecule?

6/17/15

Decided to use CikA as negative regulator/inhibitor instead of Lab A. CikA better characterized. Reviewed (Gutu. O’Shea, 2013).

6/18/15

Finished design on constructs 1, 2, 3 Deciding on RBS, perhaps need to use high efficiency promoters and lower efficiency RBS. Justin will email kaibc/Kai analog sequences. Decided on meeting 4pm Tuesday.

To Discuss: Specific RBS and promoter strengths on different genes.

Started Testing for Competent Cells

*Used Justin/Rust Lab protocol instead of iGEM/team protocol because did not have cmrR plates.

Labels:

Plasmid of Interest- PJ006, containing KaiABC under kaiA, and kaibc promoters, Spec resistance, Conc= 100ng/uL
Transformed plate- PJ006 MC 6/18/15

Steps

1. Remove cells from freezer, incubate on ice

2. Add 1 uL DNA (100ng/uL) into competent cell tube

3. Incubate tube on ice for 30 mins

4. Incubate tube 42C water bath for 1 min heat shock

5. Incubate tube ice 5 mins

6. Rescue cells by pipetting 900 uL LB into tube (use sterile flame)

*Used LB from MJR Lab, will have to make LB tomorrow

7. Incubate tube in shaker 37C for 1 hour

8. Heat Spec plate in incubator as cold plate reduces efficiency,complete while cells shaking

9. Collect pellet, spin 3000 rcf/gs for 3 mins

10. Decant 800 uL of supernatant

11. Use glass beads (5-6) per section (use sterile flame)

12. Mix pellet, pipetted 200 uL in total, 180 to 0.90 section, 20 to 0.10 section

13. Shake with beads and remove

14. Incubate plate overnight 37C

6/19/15

Checked on Transformed Plates

Good growth on both sections.

Transformation efficiency

0.10 Section

= (293 cfus) / ( ((1 uL x 100 ng/uL)/1000 uL soln)(20/200 uL plated))

= 293 cfus/0.01 ng DNA plated = 2.93 x 10^4 transformants/ng

To Discuss: How to improve Transformation Efficiency.

Made 500 mL LB Solution

Made CM plates

Temperature of Freezer= 6 C, Chloramphenicol storage temperature= 2-8 C

125 uL of 50mg/ml cm used for every 250 uL plate soln made

Poured plates

Need to label once plates set overnight

Updated restock list, will go tomorrow to buy new supplies (check Inventory)

6/20/15

Stock room closed :(, Will order things on Monday

Placed cmR plates in left cold room, bottom right corner of room behind some Rust plates

Week 2:

Goals- Finalize construct design, develop Competent Cells

6/22/15

Worked on Week 1 Presentation

Streaked 1 Tube of Comp 2014 Cells on LB Only Plate for Competent E.coli procedure -protocol under master list titled “Competent E.Coli 6/21/15”

Started Mini-prep to extract Kai proteins. Inoculated 2 colonies, 1 each in 2mL LB + Spec medium.

6/23/15

Cultured 5 colonies from LB only plate into 10 mL LB

Week 1 meeting today

Meeting Notes:

→ Discussed project direction and construct design. Need to add terminator after RpaA. Need to decide whether or not fusion protein construct worth it. Given that RpaA might have some basal phosphorylation level, induced CikA should present some results. How the constructs are set up now, ideal tests can only be conducted with all three constructs. Don’t need to perhaps mutagenize cut sites as these plasmids will not be final bio-brick. Final biobrick would possibly only have CikA, SasA. Overall consensus is that CikA worth exploring. Need to develop primers ASAP.

→ Need to develop more work on biosynthesis pathway and decide which molecule want to consider as well as what is focus of experiment. Want KaiABC as submitted biobrick so perhaps focusing on biosynthesis pathway is a bit ambitious. Need to consider perhaps alternative, simpler molecule.

→ Will likely assemble using Gibson, order this free kit from RPI.

Contact Danny for competent cells and Justin for Gibson primers

6/24/15

Worked on designing primers. Contacted Danny for competent cell procedure, will complete on Friday. Will conduct both CaCl2 and RbCl2 procedures. Already have streaked LB only plate for competent cells in cold room.

6/25/15

Finished designing primers, will check with Kevin tomorrow. Inoculated two 5mL LB broths with 1-3 colonies each. NEB Gibson Assembly Kit with competent cells arrived!

6/26/15

Carried out competent cell procedure.

http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf

https://drive.google.com/open?id=0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV

Used both Rust Lab and RbCl2 as a comparison.

Week 3:

Goals- Test efficiency of competent cells, start as much cloning as possible

6/29/15

Conducted Transformation of CaCl2 and RbCl2.

15. Remove cells from freezer, incubate tubes on ice

16. Add 1 uL DNA (50 pg//uL) into competent cell tube

17. Incubate tube on ice for 30 mins

18. Incubate tube 42C water bath for 1 min heat shock

19. Incubate tube ice 5 mins

20. Rescue cells by pipetting 850 uL LB into tube (use sterile flame)

21. Incubate tube in shaker 37C for 1 hour

22. Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking

23. Collect pellet, spin 3000 rcf/gs for 3 mins

24. Decant 800 uL of supernatant

25. Use glass beads (5-6) per section (use sterile flame)

26. Mix pellet, pipetted 200 uL in total

27. Shake with beads and remove

28. Incubate plate overnight 37C

Spec on LB negative control culture overnight =-0.019 A (no growth at all)

= (52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 uL plated))

= 52 cfus/(4.5 x 10^-5) ng DNA plated =1.15 x 10^6 transformants/ng (Rust)

=52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 uL plated))

= 18 cfus/(4.5 x 10^-5) ng DNA plated =4.00 x 10^5 transformants/ng (RbCl2)

6/30/15

Primers for first constructs arrived, however at team meeting discussed how plasmids need to be re-designed to effectively compare SasA and CikA. Also CikA will need KaiB, and likely to add RpaB to be consistent with Chen et al. Constructs for Read-Out system were revamped, and constructs for Oscillation system were designed as well.

Week 4:

Goals- Order final gBlocks and primers. Standardize and develop specific, in depth protocols. Practice Western Blots, start writing project report.

7/20/15

gBlocks for Oscillation and Read-Out systems were modified. See Dropbox for final edits and modifications.

7/21/15

gBlocks for Oscillation and Read-Out systems were finally ordered. Primers were designed.

7/22/15

Primers ordered. Reached out to grad advisers for Western Blotting techniques. Researched Gibson Assembly and Western Blot protocols. Will need to research GFP Protocols.

GFP Protocols

http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf

7/23

Materials for practice western blot acquired. Gibson Assembly protocol drafted.

7/24

Started western blot. See Rust Lab protocol. Slight changes include 7.5% gel used, cassette assembled not submerged in buffer. Primers diluted and placed in -20 fridge.

7/27

Primary and secondary antibody staining accomplished. Experiments more clearly laid out. Need to start considering plan for pRha inducible promoter and how to alter stoichiometry.

Week 5:

Goals- Finalize and outline protocols, generate explanations for plasmids and background info for wiki and presentation, order materials, gblock assembly

7/28

Western blot procedures expanded. See Aaron’s email about compatible backbones. This week discussed assays -will need to western blot for KaiA before investigating oscillatory system in order to characterize input L-Rhamnose to output Kai A production. Dilutions will occur on log scale first for L-Rhamnose.

7/29

Finished specific protocols -need to ask White lab for sonicator?. Looked up compatibility of backbones. pSB1,3,4 have pMB1 (copy number 100-300/cell), p15A (low-medium 10-12 copy), pSC101 (~5 copies/cell). Will need to construct primers for kaibc/GFP onto the SasA+CikA/SasA plasmids.

7/30/15

Materials reviewed and listed. Meeting with Barry to talk about iGEM as a class. Went over protocols and methods. Allocated who is ordering what.

7/31/15

Gibson Assembly

5 uL of Gibson HiFi Master mix was used in each assembly reaction to minimize amount reagent used. Amount of blocks used, dependent on bps of each block relative to each other. Each gblock diluted in 20 uL of dH₂O. Used standardized amount 50 ng of largest block in each assembly. Assembled on ice. Incubated on 50^oC heatblock for 1 hour.

mage 1.png

PCR

Assembled 5.5 times of 1X Master Mix (not on ice). Dilute 100 uM (100X) primers to 10X primers. 2 uL of primer and 18 uL of dH₂O. Aliquoted 49 uL of Master Mix with 1 uL from Gibson Assembly Mix.

Recipe Phusion Master Mix:

o Phusion 5X GC Buffer = 10uL x 5.5 = 55 uL

o dNTPs 10 mM = 1 x 5.5 =5.5 uL

o F Primer MC003 10 uM = 2.5 x 5.5 =13.75 uL

o R Primer MC004 10 uM = 2.5 x 5.5= 13.75 uL

o Phusion DNAP = 0.5 x 5.5 =2.75 uL

o H₂O = 32.5 x 178.75 uL

o DMSO = 1.5 x 5.5 =8.25 uL

*Should have added only 170.5 uL (31 x 5.5) –Mix slightly more dilute

Thermocycler Settings: 30 cycles, 98^o for 30s, 98^o for 10s, 65^o for 30s, 72^o for 1 min, 72^o 7 min, Hold 4^o

Week 6:

Goals- gblock assembly and Transformation

8/3/15

Decided on backbones:

Oscillator MC001 – Cmr (standard igem backbone for submitted biobrick)

Readout SasA MC002 –Amp (theoretically want to use with MC001 if successful) à need to add GFP + kai bc

Readout SasA/CikA MC003 –Amp (“ “) à need to add GFP + kaibc

KaiC Variants MC004-7- Cmr (would never use with MC001)

Agarose Gel Casting

Made 50 mL of 1% Agarose gel. General procedure: added agarose and 1X TAE into ER flask, microwaved until boil, cool under water, poured into tray, added 0.75uL EtBr for visualizing, inserted combs, cool in cold room for 15-20 mins.

Gel Electrophoresis

Loaded 10uL of 6X loading dye into 50uL samples. Loaded 10 uL of 1 kB Plus DNA Ladder from Invitrogen. Seems to be issue w/amount of sample loaded –only 15-29 uL available. Run under 120V for 30 mins. Could be issue with evaporation in thermocycler. Gel too big for tray. Yields of products exist, however seems low. Issue with MC001 –no clear product visible. PCR should be redone.

Image:

Gel Extraction

Gel Weights:

MC001 –N/A

MC004 -0.1536 g

MC005 -0.2048 g

MC006 -0.0965 g

MC007 -0.3962 g

*1mg = 1uL

Added 3x uL QG buffer to volume of gel and 1X uL of isopropanol to volume of gel.

PCR

To redo PCR for MC001,4,5,6,7, 1X Master Mix for 15 reactions made. Two reactions for each construct.

PCR Master Mix Recipe-

o Phusion 5X GC Buffer = 150 uL

o dNTPs 10 mM = 15 uL

o Phusion DNAP = 7.5 uL

o H₂O = 465 uL

o DMSO = 22.5 uL

Added 2.5uL of F and R Primers, 44 uL of Master Mix, and 1uL of DNA for each sample. Thermocycler settings- 98^o 2 min, 98^o 15 s, 69^o 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

PCR Master mix also used for amplifying linear Cmr backbones (diluted in 10uL, used 1 uL of sample for Phusion PCR).

8/4/15

Gel Electrophoresis

Made 100 mL of 1% Agarose gel. 10 uL, 1kB plus Ladder loaded. 35uL MC001, 20uL MC001, 34 uL MC004, 34 uL MC004, 33 uL of MC006,7,8 and Cmr backbones. Products from MC004,5,6,7 and Cmr Linearized backbones extracted using gel punches (borrowed from Rust Lab, need to order more to return). MC001 still not very good yield. Next step to purify MC004-7 and linearized backbones, redo Gibson and PCR of MC001 using gradient thermocycler.

Image:

Gibson Assembly

5 uL of Gibson HiFi Master mix, 1 uL PMC001_b1, 0.5965 uL PMC001_b2, 3.4305 uL H₂O, heatblock for 1 hour 50^oC.

PCR

Master mix of 70 uL created (calculate ratio of 1X x 7/5)

o Phusion 5X HC Buffer = 14 uL

o dNTPs 10 mM = 1.4 uL

o Phusion DNAP = 0.7 uL

o H₂O = 43.4 uL

o DMSO = 2.1 uL

o DNA =1.4 uL

o F Primer MC003= 3.5 uL

o R Primer MC004 =3.5 uL

10uL Master mix aliquoted into 7 samples.

Thermocycler settings- 98^o 2 min, 98^o 15 s, 60^o_,62^o, 64^o, 66^o, 68^o, 70^o, 72^o 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

Actual Anneal temperatures- 60.0^o, 62.0^o, 63.3^o, 66.6^o, 68.2^o, 69.7^o, 72.0^o

T=66.0^o, G=6.0^o for 30 cycles

8/5/15

Gel Electrophoresis

Made 90 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye). Run for 120V, 30 mins. EtBr cloud on gel seen, only ladder shows visible bands. No other bands visible. Likely error with PCR and addition of EtBr.

Image:

Gel Extraction

Used Promega spin columns/buffer to concentrate in 15 uL of DNA

Purity Yields using nanodrop - C1 (Cam Backbone) -

C2 (Cam Backbone) -58.9 ng/uL

4 - 34.7

4’ -

5 - 31.5

6 - 36.3

7 - 40.1

PCR

To redo PCR for Gibson products of MC001, 75 uL of 1X PCR Master Mix

PCR Master Mix Recipe-

o Phusion 5X HF Buffer = 15 uL

o dNTPs 10 mM = 15 uL

o Phusion DNAP = .75 uL

o H₂O = 46.5 uL

o DMSO = 2.25 uL

o DNA (products from Gibson 8/4)= 1.5uL

o F Primer MC003= 3.75uL

o R Primer MC004 =3.75uL

10 uL of Master Mix aliquoted into each sample tube.

To PCR PMC001_b1 for confirmation of block and analysis of primers

50 uL of 1XPCR Master Mix Recipe-

o Phusion 5X HF Buffer = 10 uL

o dNTPs 10 mM = 1 uL

o Phusion DNAP = .5 uL

o H₂O= 31 uL

o DMSO = 1.5 uL

o DNA (pMC001_b1)= 1 uL

o F Primer MC005= 2.5uL

o R Primer MC006 =2.5uL

Added 2.5uL of F and R Primers, 44 uL of Master Mix, and 1uL of DNA for each sample..

Thermocycler settings- 98^o 2 min, 98^o 15 s, 60^o_,62^o, 64^o, 66^o, 68^o, 70^o, 72^o 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

Actual Anneal temperatures- 60.0^o, 62.0^o, 63.3^o, 66.6^o, 68.2^o, 69.7^o, 72.0^o

T=66.0^o, G=6.0^o for 30 cycles

8/6/15

Gel Electrophoresis

Made 100 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye) and 1 50uL sample (divided into two wells, 42uL in one well 18 in the other). Run for 120V, 30 mins. No product clear enough to extract. Imaging gel shows faint products under 68,70, and 72^o. Could mean issue with primers. Strangely, no product of right gBlock size seen. Again could be primers.

Image:

Gibson Assembly:

Assembled purified biobricks MC004, MC005, MC006, MC007 into Cam backbone. Used following recipe based on bps of insert and backbone.

Transformation of Assembled products:

DNA straight from Gibson Assembly Reaction was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: 4.00 x 10^5 transformants/ng (RbCl2)

1. Remove cells from freezer, incubate tubes on ice

2. Add 1 uL DNA (50 pg//uL) into competent cell tube

3. Incubate tube on ice for 30 mins

4. Incubate tube 42C water bath for 1 min heat shock

5. Incubate tube ice 5 mins

6. Rescue cells by pipetting 900 uL LB into tube (use sterile flame)

7. Incubate tube in shaker at 37C/1100 RPM for 1 hour

8. Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking

9. Collect pellet, spin 3000 rcf/gs for 3 mins

10. Decant 800 uL of supernatant

11. Use glass beads (5-6) per section (use sterile flame)

12. Mix pellet, pipetted 200 uL in total

13. Shake with beads and remove

14. Incubate plate overnight 37C

*Negative control w/no transformed DNA resulted in 0 colonies. Negative control set up on 8/7.

PCR

PCR of 8/3 Gibson PCR, 8/4 Gibson PCR conducted for further amplification. Block 4.1 PCR as positive control. Block 1.1 and 1.2 PCR run to increase DNA amount in hopes of Gibson from amplified blocks. 50 uL of sample for each PCR (5 samples in total).

Added Ingredients to individual Samples

o Phusion 5X HC Buffer = 10 uL

o dNTPs 10 mM = 1 uL

o Phusion DNAP = 0.5 uL

o H₂O = 31.0 uL

o DMSO = 1.5 uL

o DNA =1.0 uL

o F Primer = 2.5 uL

o R Primer =2.5 uL

10uL Master mix aliquoted into 7 samples.

Thermocycler settings- 98^o 2 min, 98^o 15 s, 70^o, 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

Primers for each sample

Gibson 8/3 -MC003/MC004

Gibson 8/4 -MC003/MC004

pMC001_b1 -MC005/MC006

pMC001_b2 -MC007/MC008

pMC004_b1- MC019/MC020

*Upon further examination, should have used MC003 for pMC004_b1

8/7/15

Gel Electrophoresis

1% Agarose gel run of PCR products from 8/6. Could not see products under blue light. Under UV light, products seemed more specific. Still not as strong as in previous gels. Perhaps need to troubleshoot PCR better.

Image:

Transformation Results

Used iPhone application “ColonyCount” to assist in counting plates.

Plate Numbers are indicated on lower left of the pictures.

Average is 321 colonies.

Transformation Efficiency

Used 1ul of 50pg/ul of DNA

4: 287 / 5*10^-5 = 5.74*10^6 cfu/ug

5: 328 / 5*10^-5 = 6.56*10^6 cfu/ug

6: 414 / 5*10^-5 = 8.28*10^6 cfu/ug

7: 254 / 5*10^-5 = 5.08*10^6 cfu/ug

Primers

Designed Sequencing primers as well as new primers for pMC001. Primers made specifically for oscillator plasmid -overhangs incorporated to make primers longer and more specific.

PCR

Prepared PCR of products seen on gel in morning (G1, G2, 001b1, 001b2, 004b1, used 1 uL of leftover PCR reaction). Used Q5 High Fidelity polymerase, as no Phusion available.

Ingredients:

Q5 High-Fidelity 2X Master Mix- 25 uL

DNA -1 uL

F Primer -2.5 uL

R Primer -2.5 uL

H2O -19 uL

Thermocycler Settings: 98C 30s, 98C 10s, 65C for 30s, 72C for 30s, 72C for 2mins, hold at 4C

30 cycles

Prepared Colony PCR To confirm inserts of pMC004,5,6,7. Used Taq DNA polymerase instead of phusion.

1. Pick single colony from plate, place in 50uL of dH2O (acts as DNA template)

2. Add following PCR 1X Master Mix for Taq:

5 uL 10X buffer

1 uL dNTPs

1 uL 10 uM primer stock-VF2 and VR

1 uL DNA stock

0.5 uL Taq

41.5 uL H2O

Thermocycler Settings: 95C 2mins, 95C 15s, 55C 15s, 68C 45s (30 cycles), 68C 10m, hold 4C

-> Extend to 1min per kb (look up on product sheet)

8/9/15

Gel Electrophoresis-

Ran 1% Agarose gel 120V, 30 mins. Ran both Colony PCR and Q5 PCR. 5uL each sample loaded. Different ladder used (Quick Load Purple 2-Log from NEB. Same amount of EtBr (0.75 uL) used.

8/10/15

Gel Electrophoresis-

Gel repeated, this time using leftover 45uL of sample. 1kB Plus invitrogen ladder used.

Innoculation-

Due to failure of Colony PCR, colonies were inoculated and incubated. Will conduct direct miniprep on 8/11 and sequence to sequence. This should help in determining if there is a problem with primers/insert or with the PCR.

Gibson Assembly-

Gibson assembly of BH001_b1 and Cam backbone conducted.

PCR-

Set up PCR for biobricks MC002, and MC003 and BH001 (confirmation).

11X PCR Master Mix

PCR Master Mix Recipe-

o Phusion 5X HF Buffer = 110 uL

o dNTPs 10 mM = 11 uL

o Phusion DNAP = 5.5 uL

o H₂O = 341 uL

o DMSO = 16.5 uL

44uL of Master mix, 2.5 uL of F primer, 2.5 uL of R Primer and 1 uL of template DNA used.

DNA Template	Primers	Info	Gel to Run
MC002_b1	MC029, MC010 1815 bps	Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts	1% Agarose
MC003_b1	MC029, MC014 1815 bps	Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts	1% Agarose
MC008_b1	MC003, MC028 1294 bps	Clone out kaibc promoter/GFP	1% Agarose
Biobrick B0015 Terminator	MC027, MC030 129 bps	Clone out terminator	2% Agarose
BH001/Cam Gibson	MC003, MC004 765 bps *should not have done	? Was to clone out insert, should have transformed as is.	1% Agarose

Thermocycler settings- 98C 2min, 98C 15s, 67C 30s, 72C 1.5 mins, 72C 10min, 4 hold.

8/11/15

Gel Electrophoresis-

2% gel for Terminator cloning sample and 1% gel for other PCR samples (see table) run. 120V for 30 min.

Miniprep

BH protocol

PCR

PCR of Minipreps

(BH protocol)

PCR of MC002/3 blocks

Phusion 7 x of 1X mix

HF Buffer -70 uL

DMSO -10.5 uL

DNTPs -7 uL

H2O -219 uL

Phusion DNAP -3.5 uL

Use 44 uL of master mix w/ 2.5 of F and R primers and 1 uL of template DNA.

DNA Template	Primers
MC002_b1	MC029, MC010 1815 bps product
MC003_b1	MC029, MC014 1815 bps product
MC008_b1	MC003, MC028 1294 bps product

Thermocycler Settings-

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 65°C 72°C	10 seconds 30 seconds 1 min (30s x 1.8kb -largest product)
Final Extension	72°C	10 min
Hold	4°C	Hold

Thermocycler Settings for Mini-Prep PCR-

STEP	TEMP	TIME
Initial Denaturation	98°C	120 seconds
30 Cycles	98°C 61.6°C (NEB - DMSO%*0.8) 72°C	15 seconds 30 seconds 1 min (30s x 1.8kb -largest product)
Final Extension	72°C	5 min
Hold	4°C	Hold

8/12/15

PCR of Terminator

2 50 uL Samples

HF Buffer -10 uL

DMSO -1.5 uL

DNTPs -1 uL

H2O -31 uL

Phusion DNAP -0.5 uL

10 uM of MC027 Primer -2.5 uL

10 uM MC030 Primer -2.5 uL

DNA from plate -1 uL

Thermocycler Settings: 1 cycle: 98C for 2 mins, 5 cycles: 98C for 15s, 69C for 30s, 72C for 2 min 30 cycles: 98C for 15s, 72C for 1.5 min, 1 cycle 72C for 10 min, 4C hold.

Gel Electrophoresis-

50 mL 2% gel, 150 mL 1% gel, and 100 mL 1% gel run for Miniprep PCR as well as MC002/3 clone parts PCR. 25 uL of sample loaded for Terminator 2% gel. 19 uL sample loaded for 150mL Miniprep gel. 45 uL of sample loaded for MC002/3 clone parts 1% gel. 1kB plus Invitrogen ladder used. Send samples 41,43,51,52,53,54,61,62,63,64,74 for sequencing.

Images:

Gel Purification-

Extracted correctly sized product fragmnets under blue light: T-189 bps, GFP 1249 bps.

Weights of gels:

GFP 1-63.8 mg

GFP 2-70.64 mg

T 1-34.6 mg

T 2-110.0 mg

Used Machery-Nagel clean up method:

1. Add 200uL NTI Binding buffer / 100 mg gel

2. Incubate at 50C for 10 mins

3. Transfer solution to spin column and collection tube.

4. Spin at 11,000g (RCFs) for 30s

5. Discard flow through. Add 700 uL of wash buffer NT3 to spin column.

6. Spin at 11,000g (RCFs) for 30s

7. Discard flow through. Add 700 uL of wash buffer NT3 to spin column.

8. Spin at 11,000g (RCFs) for 30s

9. Spin at 11,000g (RCFs) for 1 min to dry silica membrane

10. Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 30s

11. Nanodrop (use EB buffer as blank, load 1.5 uL sample)

Purity recorded w/Nanodrop:

T1- 5.1ng/uL 260/280-14.84

T2-23.1ng/uL 260/280-1.92

GFP 1- 41.6ng/uL 260/280-1.94

GFP 2- 33.1ng/uL 260/280-2.03

Plan for MC002_b1, MC003_b1: There is low yield of insert probably due to repeating regions of DNA in blocks 1 of MC002 and MC003. As IDT expressed, there are a lot of low mass products that are more efficiency amplified by primers. Therefore, as Jennifer Moran mentioned, the best course of action would be to insert these two gblocks into a Zero Blunt TOPO PCR Vector and transforming into competent cells to amplify our gblocks. After producing colonies, we can PCR and then screen for colonies with correct product size. We will then miniprep and send these blocks in for sequencing. This will take more time, but will ensure the purity of the insert. After confirming the correct sequence, the DNA from the miniprep/gel extraction? can be used to gibson the GFP/kaibc, the terminator, and the first blocks together.

Mini-Prep Results (Nano-Drop)

Sample	260/280	260/230	ng/ul
41	1.86	2.20	55.1
42	1.84	2.06	35.4
43	1.89	2.10	72.9
44	1.92	1.83	30.7
51	1.94	1.87	46.8
52	1.90	2.00	53.9
53	1.90	2.15	66.6
54	1.93	2.14	58.6
61	1.97	2.06	52.2
62	1.97	2.14	61.1
63	2.00	2.24	52.5
64	1.92	2.12	57.1
71	2.03	2.13	56.9
72	1.88	1.81	50.2
73	1.93	1.83	59.3
74	1.92	1.92	53.3

The highlighted ones are the ones that we choose to sequence.

Sequencing-

The concentration of the DNA templates were too low, so we used 10 ug of each.

The primers were diluted to a 4uM solution from a 100x stock.

(1.4 ul of primer + 33.6 ul of water)

VF2 [1]

MC003 (F) [2]

MC041 (F) [3]

MC023 (F) [4]

MC022 (R) [5]

VR [6]

[41]

[43]

[53]

[54]

[62]

[63]

[71]

[73]

^Things sent.

Transformation of BH001:

DNA was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: 4.00 x 10^5 transformants/ng (RbCl2)

15. Remove cells from freezer, incubate tubes on ice

16. Add 1 uL DNA (50 pg//uL) into competent cell tube

17. Incubate tube on ice for 30 mins

18. Incubate tube 42C water bath for 1 min heat shock

19. Incubate tube ice 5 mins

20. Rescue cells by pipetting 900 uL LB into tube (use sterile flame)

21. Incubate tube in shaker at 37C/1100 RPM for 1 hour

22. Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking

23. Collect pellet, spin 3000 rcf/gs for 3 mins

24. Decant 800 uL of supernatant

25. Use glass beads (5-6) per section (use sterile flame)

26. Mix pellet, pipetted 200 uL in total

27. Shake with beads and remove

28. Incubate plate overnight 37C

8/13/15-

Transformation Efficiency of BH001-

amt dna used/plate

Plates 1 and 2 were discarded, a colony PCR was done on 6 samples from Plate 3

They were PCRed separately with two different annealing temperatures, 61.6 and 66.

Gibson Assembly

Gibson assembly using 5uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hour 50C heatblock.

PCR-

Prepared 2 50uL 1X PCR samples.

HF Buffer -10 uL

DMSO -1.5 uL

DNTPs -1 uL

H2O -31 uL

Phusion DNAP -0.5 uL

F Primer 10 uM MC003 -2.5 uL

R Primer 10uM MC004 -2.5 uL

DNA (Gibson assembly rxn 8/13) -1uL

Thermocycler Settings-

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 65°C 72°C	10 seconds 30 secondsc 1.65 mins (30s x 3.3kb -largest product)
Final Extension	72°C	10 min
Hold	4°C	Hold

Thermocycler Settings for Mini-Prep PCR- Low Temp

STEP	TEMP	TIME
Initial Denaturation	98°C	120 seconds
30 Cycles	98°C 61.6°C (NEB - DMSO%*0.8) 72°C	15 seconds 30 seconds 1 min (30s x 1.8kb -largest product)
Final Extension	72°C	5 min
Hold	4°C	Hold

Thermocycler Settings for Mini-Prep PCR- High Temp

STEP	TEMP	TIME
Initial Denaturation	98°C	120 seconds
30 Cycles	98°C 66°C 72°C	15 seconds 30 seconds 1 min (30s x 1.8kb -largest product)
Final Extension	72°C	5 min
Hold	4°C	Hold

Gel Electrophoresis-

100 mL of 1% agarose gel run 120V, 30 mins. Samples loaded include 45 uL of BH003 biobrick and 20 uL of colony PCRs. 5 uL 1 kB plus Invitrogen ladder loaded. Colony PCRs yielded no results, biobrick BH003 extracted using gel punches (seen in image). First column of BH3 seemed to give very low yield (light band not strong band seen before extraction).

Image-

Gel Extraction-

BH003 biobrick extracted from gel. Machery-Nagel protocol used for gel extraction.

Weight of gel: 85.2 mg

Used Machery-Nagel clean up method:

12. Add 200uL NTI Binding buffer / 100 mg gel

13. Incubate at 50C for 10 mins

14. Transfer solution to spin column and collection tube.

15. Spin at 11,000g (RCFs) for 30s

16. Discard flow through. Add 700 uL of wash buffer NT3 to spin column.

17. Spin at 11,000g (RCFs) for 30s

18. Discard flow through. Add 700 uL of wash buffer NT3 to spin column.

19. Spin at 11,000g (RCFs) for 30s

20. Spin at 11,000g (RCFs) for 1 min to dry silica membrane

21. Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 30s

22. Nanodrop (use EB buffer as blank, load 1.5 uL sample)

Nanodrop purity- 14.4 ng/uL, 260/280- 1.55

PCR-

Made 4 1X 50uL samples to PCR ampicillin backbone. Used 1 uL of 25ng/uL amp linearized backbone. Primers MC001 and MC002.

PCR Master Mix Recipe-

o Phusion 5X HF Buffer = 50 uL

o dNTPs 10 mM = 5 uL

o Phusion DNAP = 2.5 uL

o H₂O = 155 uL

o DMSO = 7.5 uL

o DNA (products from Gibson 8/4)= 1.0uL

o F Primer MC001= 2.5uL

o R Primer MC002 =2.55uL

Thermocycler settings:98C 30s, 25 cycles: 98C 10s, 69C 30s, 72C 1.5m, 72.0C 10 min, 4C hold

Gibson Assembly-

Given success of gibson and transformation with BH001, decided to try assemble pMC001 and transform directly. Reaction incubated on heatblock for 1 hour at 50C.

Gel Electrophoresis-

50 mL of 1% agarose gel made to run amp backbone amplifications. 0.5 uL EtBr used. Gel run 120V for 30 mins. 1 kB Plus Invitrogen ladder used. 45 uL of samples loaded.

Image-

Gel Extraction and Purification-

Ampicillin backbone extracted from gel. Machery-Nagel protocol used for gel extraction.

Weight of gel samples:

A1 -135.8 mg (271.6 uL of binding buffer NTI used)

A2 -201.4mg (402.8 uL of binding buffer NTI used)

Nanodrop concentrations of ampicillin backbone samples

A1 - 32.3 ng/uL, 260/280- 1.84

A2 - 33.4 ng/uL, 260/280- 1.59

Gibson-

Gibson assembly of ampicillin backbone to BH003 insert conducted. Used 5uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hour 50C heatblock.

Transformation of BH003:

DNA was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: 4.00 x 10^5 transformants/ng (RbCl2)

29. Remove cells from freezer, incubate tubes on ice

30. Add 1 uL DNA (50 pg//uL) into competent cell tube

31. Incubate tube on ice for 30 mins

32. Incubate tube 42C water bath for 1 min heat shock

33. Incubate tube ice 5 mins

34. Rescue cells by pipetting 900 uL LB into tube (use sterile flame)

35. Incubate tube in shaker at 37C/1100 RPM for 1 hour

36. Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking

37. Collect pellet, spin 3000 rcf/gs for 3 mins

38. Decant 800 uL of supernatant

39. Use glass beads (5-6) per section (use sterile flame)

40. Mix pellet, pipetted 200 uL in total

41. Shake with beads and remove

42. Incubate plate overnight 37C

8/14/15

Transformation-

MC001 and BH003 gave good results from transformation. Colonies were small, but evenly distributed. Colonies could be small because plates incubated late last night.

Colony PCR of BH003/MC001-

Diluted one colony from plates with most growth into 50uL of dH2O to serve as DNA template.

Made 8.5 x of 1X PCR Master Mix:

HF Buffer: 85 uL

DMSO: 12.75 uL

dNTPS: 8.5 uL

H2O: 263.5 uL

DNAP: 4.25 uL

Used 2.5uL of VF2 and VR each and 1 uL of sample DNA.

Thermocycler Settings for Colony

STEP	TEMP	TIME
Initial Denaturation	98°C	10 minutes
30 Cycles	98°C 66°C 72°C	15 seconds 30 seconds 2.5 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Sequencing Primers for MC001- MC003,MC031,MC032,MC006,MC033, MC008

8/15/

Gel Electrophoresis

1% Agarose gel run for colony PCR. 1 kB Invitrogen plus ladder used. 50 uL of sample loaded.

8/17

Sequences Submitted

MC001 note submitted on weekend for sequencing. MC001 submitted in morning for sequencing. 4uM primer stock made by diluting in water (1:24 primer:water ratio). 14 uL of miniprepped DNA submitted with 10uL of 4uM primers.

Primers used:

VF2 (1)

VR (2)

MC003 (3)

MC031 (4)

MC032 (5)

MC006 (6)

MC033 (7)

MC008 (8)

Colony PCR

Colony PCR of MC001 set up again as upon closer examination, insert seems too small to be correct.

Diluted one colony from plates with most growth into 50uL of dH2O to serve as DNA template.

Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2 uL DNA):

HF Buffer: 18.00

DMSO: 2.70 uL

dNTPS: 1.80

H2O: 55.

DNAP: 4.25 uL

Used 4.50 uL of 10X MC003 and MC004 for mix.

Thermocycler Settings for Colony

STEP	TEMP	TIME
Initial Denaturation	98°C	10 minutes
30 Cycles	98°C 66°C 72°C	15 seconds 30 seconds 2.5 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Talked with Justin about ordering materials -TOPO kit should come tomorrow.

8/18

Gel Electrophoresis-

0.8% (to separate out larger fragments with more efficiency) agarose gel cast. Gel run on 120V for 30 mins. 1kB plus Invitrogen ladder used. No results from colony PCR, indicating something wrong during PCR. Likely issue with primers? ¾ Used instead of VF2 and VR. Have double checked primers are correct -could be issue with insert and primers. Note -issues with evaporation in thermocycler, which is why samples 115,116,117,118,128 could not be loaded.

Image-

Sequencing Results

Sequencing successful for MC004,5,7. MC006 and MC007 samples probably mislabelled as sequencing indicates correct MC006 phosphomimetic is in MC007 sequence and vice versa. Otherwise, MC007,5, and 4 are all correctly sequenced. Point mutation in MC006 (labelled MC007) samples base 244 of Snapgene file. Point mutation seems to be silent (GGC to GGT, translate features lists both as glycine. Four read out KaiC variants seem good to go.

MC001 -still waiting for colony PCR/ specific primers to arrive for assembly

MC002/3 -still waiting for TOPO kit to come for transformation ligation and assembly

MC004/7 -all assembled

BH001 -assembled, sequence verify

BH002 -gblocks not arrived yet

BH003 -assembled, sequence verify

BH004 -gblocks not arrived

Zero-Blunt TOPT Ligation and Transformation

We will be ligating and transforming four samples in order to allow the bacteria to amplify blocks 1 of MC002 and MC003.

MC002_b1
MC003_b1
MC002_b1 after PCR
MC003_b1 after PCR

Ligation

1. Combine 2uL product, 1 uL provided salt solution from pTOPO kit, 2uL H20, 1 uL pCR II-Blunt-TOPO vector

2. Pipette up and down a few times to mix.

3. Incubate 5 min at room temperature.

4. No overnight incubation needed! But you can leave it overnight at 4°C if you cannot proceed with the transformation right away,

Transformation

1. Thaw one vial of Chemically Competent E. coli cells per transformation on ice.

2. Add 2μl ligation product to the thawed cells. Keep on ice for 30 mins.

3. Transfer vials to a 42°C water bath for 45 seconds. Immediately return the tubes to ice for 2 min.

4. Add 250μl room temperature SOC orLB to each vial.

5. Incubate for 1 hr at 37°C.

6. Plate 200μl cells on LB + kanamycin plates.

7. Incubate overnight at 37°C

*The vector confers resistance to kanamycin, NOT ampicillin.

pTOPO kit)

Note: if you do not have a sufficient number of tranformants in 200μl, repeat the transformation, do a short spin (20‐30 sec) to gently pellet your cells just before plating. Remove 100μl of the supernatant, resuspend the cells by pipeting up and down.

PCR

Set up PCR for amplifying MC002_b1 and MC003_b1. The products from this amplification (correct size ~1850-1970 bps) will be ligated and transformed using the TOPO zero blunt kit in order to have more specific inserts.

Set up 2 50uL 1X PCR Reactions:

dNTPS: 1uL
H2O: 31 uL
DNAP: 0.5 uL
DNA: 1 uL (gblocks MC002_b1 and MC003_b1)
DMSO: 1.5uL
HF Buffer: 10uL
F Primer: 2.5uL (MC029)
R Primer: 2.5 uL (MC010 for MC002_b1 and MC014 for MC003_b1)

Thermocycler settings for gBlock PCR

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 66°C 72°C	10 seconds 30 seconds 1.65 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Innoculation

Colonies from MC001 plates were inoculated as colony PCR results were ambiguous. Samples: 115,116,117,118,125,126,127,128.

1. Aliquot enough LB for 2mL/sample into a Falcon tube. Pipette 2mL of LB into each culture tube/sample.

2. Pipette all of the colony suspension into the culture tube as well.

3. Put in antibiotic to act as primary screening for insert -Cam 1uL, Amp 4uL (antibiotics stored in the -20 fridge)

4. Place culture tubes into shaker at 37C overnight (~16 hrs)

Gel Electrophoresis

1% agarose gel used to run gel electrophoresis for MC002_b1 and MC003_b1. Gel run under 120V for 30 mins. Gel extraction of estimated product size conducted under blue light. 5uL 1kB Plus Invitrogen ladder loaded. 50uL of samples loaded.

Image

8/19/15

Transformation Results

Bacterial lawns seen on all plates. Perhaps issue with plates or competent cells are naturally resistant to kanamycin? Will try dilutions if cells are very efficient as cells could also be very conducive to transformation.

Sequencing Results

Analyzed samples 124 and 121 using Blast and chromatograms. Both samples did not have whole insert -MC031 gave no results for both samples. In both samples sequencing only at end of insert (part of KaiC w/KaiB and suffix) resulted.

Miniprep-

Miniprep inoculations 115,116,117,118,125,126,127,128.

1. Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all medium.

2. Resuspend cells by adding 250uL of resuspension buffer R3 with RNase A. Mix up and down until homogenous.

3. Add 250uL of Lysis buffer (L7) to lyse cells. Mix gently by inverting capped tube.Do not vortex, incubate at room temp for 5 mins.

4. Add 350uL of precipitation buffer N4. mix immediately by inverting tube or vigorously shaking. Do not vortex, centrifuge at 20,000 rcfs for 10 mins.

5. Load supernatant (750 uL) into Spin column (machery nagel used) and collection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow through and place column back in wash tube.

6. Add 500 uL of wash buffer W10 with ethanol. Incubate at room temp for 1 min, centrifuge column at 12,000gs for 1 min. Discard flow through. (*Optional Wash step)

7. Add 700 uL of wash buffer W9 with ethanol. centrifuge column at 12,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs. Discard flow through.

8. Place spin column into microfuge tube. Add 50uL of TE Buffer to center of column. Incubate column in room temp for 1 min.

9. Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop DNA. Store at 4C short term, -20C long term.

Nanodrop results-

115: 91.8 ng/uL, 260/280: 1.90

116: 172.0 ng/uL, 260/280: 1.82

117: 152.0 ng/uL, 260/280: 1.91

118: 120.4 ng/uL, 260/280: 1.81

125: 187.0 ng/uL, 260/280: 1.88

126: 149.2 ng/uL, 260/280: 1.88

127: 211.0 ng/uL, 260/280: 1.87

128: 222.8 ng/uL, 260/280: 1.89

PCR

Set up PCR for both miniprep PCR and PCR of MC002_b1 and MC003_b1 to redo ligation and transformation.

Set up 2 50uL 1X PCR Reactions for MC002_b1 and MC003_b1:

dNTPS: 1uL
H2O: 31 uL
DNAP: 0.5 uL
DNA: 1 uL (gblocks MC002_b1 and MC003_b1)
DMSO: 1.5uL
HF Buffer: 10uL
F Primer: 2.5uL (MC029)
R Primer: 2.5 uL (MC010 for MC002_b1 and MC014 for MC003_b1)

Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2 uL DNA):

HF Buffer: 18.00

DMSO: 2.70 uL

dNTPS: 1.80

H2O: 55.

DNAP: 4.25 uL

Used 4.50 uL of 10X MC003 and MC004 for mix.

Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 67°C 72°C	10 seconds 30 seconds 1:40 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Gel Electrophoresis-

1% agarose gel run at 120V for 30 mins. Loaded 10uL and 45 uL for minipreps and MC002/3 gel extractions respectively. Loaded 5uL of 1kB Plus Invitrogen ladder.

Image-

8/20

Sequences Submitted

MC001 samples submitted in morning for sequencing. 4uM primer stock made by diluting in water (1:24 primer:water ratio). 10 uL of miniprepped DNA submitted with 10uL of 4uM primers. Samples 115,117, 125, 127, 128 sent.

Primers used:

VF2 (1)

MC003 (2)

MC031 (3)

MC032 (4)

MC006 (5)

MC033 (6)

MC008 (7)

VR (8)

Gibson Assembly

New primers for MC001 arrived. Proceeded with Gibson assesmbly. Incubated mix at 50C for 1 hour.

PCR

Set up PCR for MC002_b1, MC003_b1, MC001_b1, MC001_b2, and MC001 Gibson products. Total 7 reaction (MC002/3 block 1s sampled twice as one will be used for gel extraction then TOPO ligation/transformation, the other will be used to amplify a second time).

Phusion 7.5 x of 1X mix

HF Buffer -75.00 uL

DMSO -11.25 uL

DNTPs -7.50 uL

H2O -232.50 uL

Phusion DNAP -3.75 uL

Add 44uL of PCR Mix to 2.5 of F and R Primer each and 1 uL of DNA

DNA : Primers

MC002_b1 : MC029, MC010

MC003_b1 : MC029, MC014

Gibson rxn : 1b1F, 1b2R

MC001_b1 : 1b1F, 1b1R

MC002_b2: 1b2F, 1b2R

Thermocycler settings:

Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 66°C 72°C	10 seconds 30 seconds 1:30 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Gel Electrohporesis-

1% Agarose gel run at 120V for 30 mins. 45 uL of samples loaded. Hard to see bands under blue light. Used UV light for quick clarification of size. No products from MC001 Gibson seen. MC001_b1 and MC001_b2 extracted separately.

8/21

PCR

Due to failure of yesterday’s PCR, gradient PCR was set up in order to determine optimal anneal temperature.

Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)

o Phusion 5X HF Buffer = 15 uL

o dNTPs 10 mM = 1.5 uL

o Phusion DNAP = 0.75 uL

o H₂O = 46.5 uL

o DMSO = 2.25 uL

o DNA =1.5 uL

o F Primer MC003= 3.75 uL

o R Primer MC004 =3.75 uL

10uL Master mix aliquoted into 7 samples.

Thermocycler settings- 98^o 2 min, 98^o 15 s, 60^o_,62^o, 64^o, 66^o, 68^o, 70^o, 72^o 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

Actual Anneal temperatures- 60.0^o, 62.0^o, 63.3^o, 66.6^o, 68.2^o, 69.7^o, 72.0^o

T=66.0^o, G=6.0^o for 30 cycles

Gel Purification

MC001_b1 and MC001_b2 extracted yesterday were purified. Used Machery-Nagel protocol.

Weights-

1b1 -13.4 mg

1b2 - 133.4 mg

Nanodrop results-

1b1 - 13.1 ng/uL

1b2 -19.2 ng/ul

Gel Electrophoresis

1% agarose gel run for gradient PCR. 120V for 30 mins. 1kB Invitrogen ladder used. 10uL of sample loaded. Expected product size ~3000 bps. No expected product size appeared on gel. Perhaps indicates issues with Gibson assembly. 24 inoculated colony PCRs also run on separate gel. 3 colonies chosen for inoculation and miniprep: 3,9 and 22.

Image-

Gibson-

Gibson of MC001 repeated. Incubated mix at 50C for 1 hour.

PCR-

PCR set up for MC001 blocks and MC001 8/21 Gibson. 6 samples in total. 1X PCR Mix for each sample set up:

DMAP: 0.5 uL

DMSO: 1.5 uL

dNTPS: 1 uL

DNA: 1uL

HF Buffer: 10 uL

H2O: 31 uL

F Primer: 2.5 uL (10uM)

R Primer: 2.5 uL (10uM)

DNA (Primers): MC001_b1 (MC001_b1_F_new, MC001_b1_R_new), MC001_b1 (MC001_b2_F_new, MC001_b2_R_new), MC001 Gibson (MC001_b1_F_new, MC001_b2_R_new)

PCR Conducted one at high anneal temperature (68C) one at low anneal temperature (64C)

Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 68 or 64°C 72°C	10 seconds 30 seconds 1:30 mins
Final Extension	72°C	10 min
Hold	4°C	Hold

Gel Electrophoresis-

1% agarose gel run at 120V for 30 mins. 1kB Plus invitrogen ladder used. 50uL of samples loaded. Results very inconclusive.

Image-

8/24

Miniprep-

Inoculated TOPO MC002_b1 and MC003_b1 samples miniprepped using invitrogen protocol.

Miniprep inoculations 115,116,117,118,125,126,127,128.

10. Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all medium.

11. Resuspend cells by adding 250uL of resuspension buffer R3 with RNase A. Mix up and down until homogenous.

12. Add 250uL of Lysis buffer (L7) to lyse cells. Mix gently by inverting capped tube.Do not vortex, incubate at room temp for 5 mins.

13. Add 350uL of precipitation buffer N4. mix immediately by inverting tube or vigorously shaking. Do not vortex, centrifuge at 20,000 rcfs for 10 mins.

14. Load supernatant (750 uL) into Spin column (machery nagel used) and collection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow through and place column back in wash tube.

15. Add 500 uL of wash buffer W10 with ethanol. Incubate at room temp for 1 min, centrifuge column at 12,000gs for 1 min. Discard flow through. (*Optional Wash step)

16. Add 700 uL of wash buffer W9 with ethanol. centrifuge column at 12,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs. Discard flow through.

17. Place spin column into microfuge tube. Add 50uL of TE Buffer to center of column. Incubate column in room temp for 1 min.

18. Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop DNA. Store at 4C short term, -20C long term.

Nanodrop results-

A21:

A22:

A31:

A32:

B21:

B22:

B31:

B32:

None were sufficient enough to send for sequencing. Likely errors also due to overgrowth of colonies.

PCR

Gradient PCR of only MC001 blocks conducted.

Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)

o Phusion 5X HF Buffer = 37.5 uL

o dNTPs 10 mM = 3.75 uL

o Phusion DNAP = 1.875 uL

o H₂O = 116.25 uL

o DMSO = 5.625 uL

o DNA =3.75 uL

o F Primer MC003= 9.375 uL

o R Primer MC004 =9.375 uL

10uL Master mix aliquoted into 7 samples.

Thermocycler settings- 98^o 2 min, 98^o 15 s, 60^o_,62^o, 64^o, 66^o, 68^o, 70^o, 72^o 30 s, 72^o 1 min, 72^o 10 min, 4^o hold.

Actual Anneal temperatures- 60.0^o, 62.0^o, 63.3^o, 66.6^o, 68.2^o, 69.7^o, 72.0^o

T=66.0^o, G=6.0^o for 30 cycles

Gel Electrophoresis

1 % agarose gel run for 30 mins at 120V. 25 uL of samples loaded. 1kB plus Invitrogen ladder loaded. Only sufficient product seen for block 2. Indicates issues with block 1.

Image

8/25

Sequencing Results: Found a sample (127) containing entire MC001 insert!

Innoculated sample 127 and prepared for induction with L-Rhamnose.

Re-plated TOPO reactions for MC002/3 as overgrowth of colonies on previous plates.

Set up inductions however, cultured at 37C instead of 30C. Pay attention to this, this is important, all the protocols have induced L-Rhamnose at 30C

8/26

Start over the inductions, follow the igem protocol file for help. You will need to make more LB. I have already made a few glycerol stocks. You will need to set up a 2mL innoculation in a culture tube as well as a 40mL innoculation in a flask. The 40mL innoculation will be used for inductions. [Finished]

Redo TOPO transformations.

8/27

8/28

8/31

Reviewed work done over past three days. Discussed ways to resolve western blot issue. Seems to be a lot of non-specific binding -we see blots of many different sizes. Blots of antibodied products are very bright relative to ladder. Kevin mentioned degradation could be an issue (would cause lots of different sizes in blots). On closer examination, 0% rhamnose has no (barely any) protein. Updated notebook, e-mailed Justin and Kevin for trouble shooting.

BH- Type up lysing assay and bradford assay -give some specifics about western blot (how much each sample, and calculations)

E-mail sleep people

9/1

Induction Solutions

In order to re-do induction western blots, made new 10% Rhamnose w/v solution by diluting 1 g of Rhamnose in 9 mL of water to bring final volume to 10mL.

Culture

Set up two 40 mL cultures of sample 127 in 250 mL flasks. Added 20uL of Cam in each, 40 mL of LB and 10uL of 2 mL sample 127 overnight culture. Note: 2mL overnight culture has been stored in room temperature for past four days -may also have to start from new glycerol stock. 40mL cultures placed in 37C incubator at 11:50.

9/2

Dilutions of Rhamnose Inducer Samples

As discussed in meetings, errors with previous western blot likely to be caused due to overexpression of pRhamnose. iGEM team used low copy number plasmid. Re-made inducer solutions using 1/10 of % from 0.001 - 0.1% in order to test lower gradient. Induction started at 2:30 am when OD600 of 40 mL innoculations was 1.6(?)

Minimal Media

Ordered M9 minimal media 5X salts on Rust Lab tab. Should take till friday to arrive.

Restriction Digest

Protocol from iGEM.

Digest

§ Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)

§ 5 ul NEB Buffer 2 (use CutSmart)

§ 0.5 ul BSA

§ 0.5 ul EcoRI-HF

§ 0.5 ul PstI

§ 0.5 ul DpnI (Used to digest any template DNA from production)

§ 18 ul dH20

§ Digest Plasmid Backbone

§ Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)

§ Add 4 ul of Enzyme Master Mix

§ Digest 37C/30 min, heat kill 80C/20 min

Ligation

§ Add 2ul of digested plasmid backbone (25 ng)

§ Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase

§ Add 0.5 ul T4 DNA ligase

§ Add water to 10 ul

§ Ligate 16C/30 min, heat kill 80C/20 min

§ Transform with 1-2 ul of product

Restreaked TOPO plate

Plates of successful TOPO reactions for MC002_b1 and MC003_b1. The colonies on these plates had overgrown -so in an attempt to isolate one colony for miniprepping, these colonies were streaked onto new Cam plates.

Transformed new TOPO reactions for MC02/3 and BH002/3

New TOPO reactions were conducted in parallel with the streaking.

Set up Gibson of BH002/3

BH002 and BH003 were gibsonsed with their respective Amp backbones and Cam backbones.

9/3

Lysed cells after induction

Cells were lysed after 10 hours of induction. Cells resuspened in lysis buffer, transferred to microfuge tube with 0.1 mL glass beads, vortex for 30 s and incuabted on ice for 1 min (repeated 5 times).

Bradford Assay

Bradford assay conducted to quanity amount of protein added. 1 mL 1x bio-rad protein reagent and 0.2 uL of protein sample used for each spectrophotometer (595 nm) reading. BSA protein standards with fixed concentrations made and absorbance measured for generating standard curve (concentration vs absorbance).

Conducted Western blot

SDS-Page gel

SDS-Page gel set up with purified proteins, cyanobacterial lysate and ecoli vector only lysate (not used in this specific assay). Proteins purified and loaded on 14-20% gel in 2 fold diluations from 100 ng to 1.56 ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins. Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins.

Transfer

Transfer constructed with membrane sandwich -plastic latched case, two whatman papers, two sponges, membrane, gel. Transfer apparatus run for 1.5 hours at 90 V.

Blocking and Staining

Memabrane blocked with 2% dry milk and TBST for 1 hour. Membrane pieces stained with primary Kai rabbit antibodies for 1.5 hours. Membrane washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane stained with secondary goat antibody for 1 hour. Membrane peices washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane imaged.

Ran colony PCR of TOPO reactions

Only MC003 TOPO worked. BH002/3 Gibsons worked, no TOPO results for MC002, BH002, BH003.

Inoculated negative control

Ecoli with vector (Cam backbone) innoculated at 37C. 2 mL innoculation generated.

Image

KaiB and KaiA gave no clear results.

9/4/15

Set up Rhamnose time course experiment and rhamnose gradient

Rhamnose time course experiment conducted in both M9 supplemented media (4% glycerol, 0.1% casamino acids, antibiotic) and LB. 40 mL culture grown overnight. Cells pelleted, washed with 1 mL water, pelleted then reintroduced into Lb or M9 media. Time course consisted of induction at 30C for 10 hours. 0.5% Rhamnose used. Time course experiment conducted in order to examine behavior of KaiA throughout induction. 2 mL of cells frozen every 2 hours for 16 hours. Shocks also conducted for 1 hour and 6 hours. Shocks conducted to emulate synchronization method -incubation in M9 media with no supplements to limit ATP available to cells. Samples after shock collected at 1 hour, 6 hours, and 62 hours.

Rhamnose gradient assay also conducted, using smaller gradient with 10 fold dilution. Induction conducted for 10 hours.

9/7/15

MC003, BH002, and BH003 colonies were submitted for sequencing in UChicago sequencing facility with respective primers. MC003 used specific primers, BH002/3 used M13 and M14 primers as they were in TOPO vector.

9/8/15

Bradford Assay

Bradford assay conducted to quanity amount of protein added. 1 mL 1x bio-rad protein reagent and 0.2 uL of protein sample used for each spectrophotometer (595 nm) reading. BSA protein standards with fixed concentrations made and absorbance measured for generating standard curve (concentration vs absorbance).

Conducted Western blot

SDS-Page gel set up with purified proteins, cyanobacterial lysate and ecoli vector only lysate. Proteins purified and loaded on 14-20% gel in 2 fold diluations from 100 ng to 1.56 ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins. Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins. Kaleidoscope ladder used.

Transfer constructed with membrane sandwich -plastic latched case, two whatman papers, two sponges, membrane, gel. Transfer apparatus run for 1.5 hours at 90 V.

SDS-Page gel

SDS-Page gel set up with purified proteins, cyanobacterial lysate and ecoli vector only lysate (not used in this specific assay). Proteins purified and loaded on 14-20% gel in 2 fold diluations from 100 ng to 1.56 ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins. Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins.

9/9

Blocking and Staining

Membrane blocked with 2% dry milk and TBST for 1 hour. Membrane pieces stained with primary Kai rabbit antibodies for 1.5 hours. Membrane washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane stained with secondary goat antibody for 1 hour. Membrane pieces washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane imaged. Blot only conducted for KaiA and KaiC as issues with obtaining KaiB antibodies.

9/10

Induction of Rhamnose with lower gradient started again. Finished western blot for Kai B from previous assay. Ran PCR of block MC002_b1, MC003_b1. Gel extracted blocks.

9/11

Bradford Assay

Bradford assay conducted to quanity amount of protein added. 1 mL 1x bio-rad protein reagent and 0.2 uL of protein sample used for each spectrophotometer (595 nm) reading. BSA protein standards with fixed concentrations made and absorbance measured for generating standard curve (concentration vs absorbance).

Conducted Western blot

SDS-Page gel

SDS-Page gel set up with purified proteins, cyanobacterial lysate and ecoli vector only lysate (not used in this specific assay). Proteins purified and loaded on 14-20% gel in 2 fold diluations from 100 ng to 1.56 ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins. Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins.

Transfer

Transfer constructed with membrane sandwich -plastic latched case, two whatman papers, two sponges, membrane, gel. Transfer apparatus run for 1.5 hours at 90 V.

Ran another PCR of MC002_b1, MC003_b1

Ran PCR of gel extracted blocks MC002_b1 and MC003_b1

9/12

Blocking and Staining

Ponceau staining conducted -membranes incubated in ponceau reagent for 5 mins. Could see faint bands for KaiA and KaiC samples. Membrane pieces blocked with 2% milkd and TBST overnight.

9/13

No primary antibody available at time, continued to block.

9/14

Membrane pieces stained with primary Kai rabbit antibodies for 1.5 hours. Membrane washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane stained with secondary goat antibody for 1 hour. Membrane pieces washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane imaged. Only kai B showed up in image.

9/15

Induction

Induction repeated. Cells pelleted, washed with 1 mL water, pelleted then reintroduced into Lb or M9 media. Concentrations:

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+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Classic 4"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Colorful 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Colorful 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Colorful 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Columns 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Columns 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Columns 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Columns 4"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Columns 5"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 4"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 5"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 6"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 7"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Grid 8"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 4"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 5"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 6"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 7"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table List 8"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table 3D effects 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table 3D effects 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table 3D effects 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Contemporary"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Elegant"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Professional"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Subtle 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Subtle 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Web 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Web 2"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Web 3"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Balloon Text"/>
+  <w:LsdException Locked="false" Priority="59" Name="Table Grid"/>
+  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+   Name="Table Theme"/>
+  <w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/>
+  <w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/>
+  <w:LsdException Locked="false" SemiHidden="true" Name="Revision"/>
+  <w:LsdException Locked="false" Priority="34" QFormat="true"
+   Name="List Paragraph"/>
+  <w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/>
+  <w:LsdException Locked="false" Priority="30" QFormat="true"
+   Name="Intense Quote"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 4"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 4"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 4"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 4"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 4"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 4"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 4"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 5"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 5"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 5"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 5"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 5"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 5"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 5"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 5"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 5"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 5"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 5"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 5"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 5"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 5"/>
+  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 6"/>
+  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 6"/>
+  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 6"/>
+  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 6"/>
+  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 6"/>
+  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 6"/>
+  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 6"/>
+  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 6"/>
+  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 6"/>
+  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 6"/>
+  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 6"/>
+  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 6"/>
+  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 6"/>
+  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 6"/>
+  <w:LsdException Locked="false" Priority="19" QFormat="true"
+   Name="Subtle Emphasis"/>
+  <w:LsdException Locked="false" Priority="21" QFormat="true"
+   Name="Intense Emphasis"/>
+  <w:LsdException Locked="false" Priority="31" QFormat="true"
+   Name="Subtle Reference"/>
+  <w:LsdException Locked="false" Priority="32" QFormat="true"
+   Name="Intense Reference"/>
+  <w:LsdException Locked="false" Priority="33" QFormat="true" Name="Book Title"/>
+  <w:LsdException Locked="false" Priority="37" SemiHidden="true"
+   UnhideWhenUsed="true" Name="Bibliography"/>
+  <w:LsdException Locked="false" Priority="39" SemiHidden="true"
+   UnhideWhenUsed="true" QFormat="true" Name="TOC Heading"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark"/>
+  <w:LsdException Locked="false" Priority="51" Name="Grid Table 6 Colorful"/>
+  <w:LsdException Locked="false" Priority="52" Name="Grid Table 7 Colorful"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 1"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 1"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 1"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 1"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 1"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 1"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 1"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 2"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 2"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 2"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 2"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 2"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 2"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 2"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 3"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 3"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 3"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 3"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 3"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 3"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 3"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 4"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 4"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 4"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 4"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 4"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 4"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 4"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 5"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 5"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 5"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 5"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 5"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 5"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 5"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="Grid Table 1 Light Accent 6"/>
+  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 6"/>
+  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 6"/>
+  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 6"/>
+  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 6"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="Grid Table 6 Colorful Accent 6"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="Grid Table 7 Colorful Accent 6"/>
+  <w:LsdException Locked="false" Priority="46" Name="List Table 1 Light"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark"/>
+  <w:LsdException Locked="false" Priority="51" Name="List Table 6 Colorful"/>
+  <w:LsdException Locked="false" Priority="52" Name="List Table 7 Colorful"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 1"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 1"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 1"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 1"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 1"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 1"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 1"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 2"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 2"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 2"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 2"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 2"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 2"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 2"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 3"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 3"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 3"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 3"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 3"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 3"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 3"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 4"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 4"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 4"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 4"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 4"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 4"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 4"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 5"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 5"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 5"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 5"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 5"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 5"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 5"/>
+  <w:LsdException Locked="false" Priority="46"
+   Name="List Table 1 Light Accent 6"/>
+  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 6"/>
+  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 6"/>
+  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 6"/>
+  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 6"/>
+  <w:LsdException Locked="false" Priority="51"
+   Name="List Table 6 Colorful Accent 6"/>
+  <w:LsdException Locked="false" Priority="52"
+   Name="List Table 7 Colorful Accent 6"/>
+ </w:LatentStyles>
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+	{font-family:Times;
+	panose-1:2 2 6 3 5 4 5 2 3 4;
+	mso-font-charset:0;
+	mso-generic-font-family:roman;
+	mso-font-pitch:variable;
+	mso-font-signature:-536858881 -1073711037 9 0 511 0;}
+@font-face
+	{font-family:"Lucida Grande";
+	mso-font-charset:0;
+	mso-generic-font-family:auto;
+	mso-font-pitch:variable;
+	mso-font-signature:-520090897 1342218751 0 0 447 0;}
+@font-face
+	{font-family:Garamond;
+	panose-1:2 2 4 4 3 3 1 1 8 3;
+	mso-font-charset:0;
+	mso-generic-font-family:roman;
+	mso-font-pitch:variable;
+	mso-font-signature:647 0 0 0 159 0;}
+@font-face
+	{font-family:"\@MS Mincho";
+	panose-1:2 2 6 9 4 2 5 8 3 4;
+	mso-font-charset:128;
+	mso-generic-font-family:roman;
+	mso-font-format:other;
+	mso-font-pitch:fixed;
+	mso-font-signature:0 134676480 16 0 131072 0;}
+ /* Style Definitions */
+ p.MsoNormal, li.MsoNormal, div.MsoNormal
+	{mso-style-unhide:no;
+	mso-style-qformat:yes;
+	mso-style-parent:"";
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-pagination:widow-orphan;
+	font-size:12.0pt;
+	font-family:"Cambria",serif;
+	mso-ascii-font-family:Cambria;
+	mso-ascii-theme-font:minor-latin;
+	mso-fareast-font-family:"MS Mincho";
+	mso-fareast-theme-font:minor-fareast;
+	mso-hansi-font-family:Cambria;
+	mso-hansi-theme-font:minor-latin;
+	mso-bidi-font-family:"Times New Roman";
+	mso-bidi-theme-font:minor-bidi;}
+h2
+	{mso-style-priority:9;
+	mso-style-unhide:no;
+	mso-style-qformat:yes;
+	mso-style-link:"Heading 2 Char";
+	mso-margin-top-alt:auto;
+	margin-right:0in;
+	mso-margin-bottom-alt:auto;
+	margin-left:0in;
+	mso-pagination:widow-orphan;
+	mso-outline-level:2;
+	font-size:18.0pt;
+	font-family:"Times",serif;
+	mso-bidi-font-family:"Times New Roman";
+	mso-bidi-theme-font:minor-bidi;
+	font-weight:bold;}
+h3
+	{mso-style-priority:9;
+	mso-style-unhide:no;
+	mso-style-qformat:yes;
+	mso-style-link:"Heading 3 Char";
+	mso-margin-top-alt:auto;
+	margin-right:0in;
+	mso-margin-bottom-alt:auto;
+	margin-left:0in;
+	mso-pagination:widow-orphan;
+	mso-outline-level:3;
+	font-size:13.5pt;
+	font-family:"Times",serif;
+	mso-bidi-font-family:"Times New Roman";
+	mso-bidi-theme-font:minor-bidi;
+	font-weight:bold;}
+a:link, span.MsoHyperlink
+	{mso-style-noshow:yes;
+	mso-style-priority:99;
+	color:blue;
+	text-decoration:underline;
+	text-underline:single;}
+a:visited, span.MsoHyperlinkFollowed
+	{mso-style-noshow:yes;
+	mso-style-priority:99;
+	color:purple;
+	text-decoration:underline;
+	text-underline:single;}
+p
+	{mso-style-priority:99;
+	mso-margin-top-alt:auto;
+	margin-right:0in;
+	mso-margin-bottom-alt:auto;
+	margin-left:0in;
+	mso-pagination:widow-orphan;
+	font-size:10.0pt;
+	font-family:"Times",serif;
+	mso-fareast-font-family:"MS Mincho";
+	mso-fareast-theme-font:minor-fareast;
+	mso-bidi-font-family:"Times New Roman";}
+p.MsoAcetate, li.MsoAcetate, div.MsoAcetate
+	{mso-style-noshow:yes;
+	mso-style-priority:99;
+	mso-style-link:"Balloon Text Char";
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-pagination:widow-orphan;
+	font-size:9.0pt;
+	font-family:"Lucida Grande";
+	mso-fareast-font-family:"MS Mincho";
+	mso-fareast-theme-font:minor-fareast;}
+span.Heading2Char
+	{mso-style-name:"Heading 2 Char";
+	mso-style-priority:9;
+	mso-style-unhide:no;
+	mso-style-locked:yes;
+	mso-style-link:"Heading 2";
+	mso-ansi-font-size:18.0pt;
+	mso-bidi-font-size:18.0pt;
+	font-family:"Times",serif;
+	mso-ascii-font-family:Times;
+	mso-hansi-font-family:Times;
+	font-weight:bold;}
+span.Heading3Char
+	{mso-style-name:"Heading 3 Char";
+	mso-style-priority:9;
+	mso-style-unhide:no;
+	mso-style-locked:yes;
+	mso-style-link:"Heading 3";
+	mso-ansi-font-size:13.5pt;
+	mso-bidi-font-size:13.5pt;
+	font-family:"Times",serif;
+	mso-ascii-font-family:Times;
+	mso-hansi-font-family:Times;
+	font-weight:bold;}
+span.apple-tab-span
+	{mso-style-name:apple-tab-span;
+	mso-style-unhide:no;}
+span.BalloonTextChar
+	{mso-style-name:"Balloon Text Char";
+	mso-style-noshow:yes;
+	mso-style-priority:99;
+	mso-style-unhide:no;
+	mso-style-locked:yes;
+	mso-style-link:"Balloon Text";
+	mso-ansi-font-size:9.0pt;
+	mso-bidi-font-size:9.0pt;
+	font-family:"Lucida Grande";
+	mso-ascii-font-family:"Lucida Grande";
+	mso-hansi-font-family:"Lucida Grande";
+	mso-bidi-font-family:"Lucida Grande";}
+span.SpellE
+	{mso-style-name:"";
+	mso-spl-e:yes;}
+span.GramE
+	{mso-style-name:"";
+	mso-gram-e:yes;}
+.MsoChpDefault
+	{mso-style-type:export-only;
+	mso-default-props:yes;
+	font-size:12.0pt;
+	mso-ansi-font-size:12.0pt;
+	mso-bidi-font-size:12.0pt;
+	font-family:"Cambria",serif;
+	mso-ascii-font-family:Cambria;
+	mso-ascii-theme-font:minor-latin;
+	mso-fareast-font-family:"MS Mincho";
+	mso-fareast-theme-font:minor-fareast;
+	mso-hansi-font-family:Cambria;
+	mso-hansi-theme-font:minor-latin;
+	mso-bidi-font-family:"Times New Roman";
+	mso-bidi-theme-font:minor-bidi;}
+@page WordSection1
+	{size:8.5in 11.0in;
+	margin:1.0in 1.25in 1.0in 1.25in;
+	mso-header-margin:.5in;
+	mso-footer-margin:.5in;
+	mso-paper-source:0;}
+div.WordSection1
+	{page:WordSection1;}
+ /* List Definitions */
+ @list l0
+	{mso-list-id:127214036;
+	mso-list-template-ids:-469198718;}
+@list l0:level1
+	{mso-level-start-at:29;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l1
+	{mso-list-id:180552230;
+	mso-list-template-ids:801819568;}
+@list l1:level1
+	{mso-level-start-at:10;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l2
+	{mso-list-id:370422956;
+	mso-list-template-ids:-189356692;}
+@list l2:level1
+	{mso-level-start-at:7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l3
+	{mso-list-id:372269068;
+	mso-list-template-ids:-855630330;}
+@list l4
+	{mso-list-id:595599278;
+	mso-list-template-ids:-93938326;}
+@list l5
+	{mso-list-id:596013480;
+	mso-list-template-ids:259270176;}
+@list l6
+	{mso-list-id:719940265;
+	mso-list-template-ids:1546034494;}
+@list l7
+	{mso-list-id:768237456;
+	mso-list-template-ids:-1302444320;}
+@list l7:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l7:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l7:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l7:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8
+	{mso-list-id:827137095;
+	mso-list-template-ids:812008446;}
+@list l8:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l8:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l8:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l8:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l9
+	{mso-list-id:965038246;
+	mso-list-template-ids:-1951135132;}
+@list l9:level1
+	{mso-level-start-at:12;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l10
+	{mso-list-id:1186166131;
+	mso-list-template-ids:336604606;}
+@list l10:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l10:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l10:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l10:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l11
+	{mso-list-id:1260065823;
+	mso-list-template-ids:844535482;}
+@list l11:level1
+	{mso-level-start-at:15;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l12
+	{mso-list-id:1355964867;
+	mso-list-template-ids:-1602552582;}
+@list l12:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l12:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l12:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l12:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13
+	{mso-list-id:1417703992;
+	mso-list-template-ids:646868248;}
+@list l13:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l13:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l13:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l13:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l14
+	{mso-list-id:1457674845;
+	mso-list-template-ids:-186115930;}
+@list l14:level1
+	{mso-level-start-at:15;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;}
+@list l15
+	{mso-list-id:1472559901;
+	mso-list-template-ids:-1190888036;}
+@list l15:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l15:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l15:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l15:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l16
+	{mso-list-id:1728990472;
+	mso-list-template-ids:-881837260;}
+@list l17
+	{mso-list-id:1803766249;
+	mso-list-template-ids:1705149868;}
+@list l17:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0B7;
+	mso-level-tab-stop:.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Symbol;}
+@list l17:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:o;
+	mso-level-tab-stop:1.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:"Courier New";
+	mso-bidi-font-family:"Times New Roman";}
+@list l17:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:1.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:2.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:3.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.0in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l17:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\F0A7;
+	mso-level-tab-stop:4.5in;
+	mso-level-number-position:left;
+	text-indent:-.25in;
+	mso-ansi-font-size:10.0pt;
+	font-family:Wingdings;}
+@list l18
+	{mso-list-id:1836604015;
+	mso-list-template-ids:1248471958;}
+@list l19
+	{mso-list-id:1889951205;
+	mso-list-template-ids:-641566644;}
+@list l20
+	{mso-list-id:1913932245;
+	mso-list-template-ids:-2022522526;}
+@list l21
+	{mso-list-id:2075807830;
+	mso-list-template-ids:1416373302;}
+@list l2:level1 lfo4
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo5
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo6
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo7
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo8
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo9
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo10
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l2:level1 lfo11
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo12
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo13
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo14
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo15
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo16
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo17
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo18
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo19
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo20
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo21
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo22
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo23
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo24
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l11:level1 lfo25
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo29
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo30
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo31
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo32
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo33
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo34
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo35
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo36
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo37
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo38
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo39
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo40
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo41
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l14:level1 lfo42
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo43
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo44
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo45
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo46
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo47
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo48
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo49
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo50
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo51
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo52
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l9:level1 lfo53
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l0:level1 lfo54
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l0:level1 lfo55
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l0:level1 lfo56
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
+	text-indent:0in;}
+@list l0:level1 lfo57
+	{mso-level-start-at:0;
+	mso-level-numbering:continue;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	margin-left:0in;
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-<!-- autogen start -->
+<body lang=EN-US link=blue vlink=purple style='tab-interval:.5in'>
-<p><b>GeneHackers Summer 2015 Journal</b></p>
+<div class=WordSection1>
-</html>
+<p class=MsoNormal align=center style='text-align:center'><span class=SpellE><b><span
-Original [[File:UChicagoLabNotebook.pdf]]
+style='font-size:26.0pt;font-family:"Calibri",sans-serif;mso-ascii-theme-font:
-<html>
+major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial;
+color:#C00000;mso-bidi-font-style:italic'>UChicago</span></b></span><b><span
+style='font-size:26.0pt;font-family:"Calibri",sans-serif;mso-ascii-theme-font:
+major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial;
+color:#C00000;mso-bidi-font-style:italic'> Summer 2015 Lab Journal</span></b><span
+style='font-size:26.0pt;font-family:"Calibri",sans-serif;mso-ascii-theme-font:
+major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:"Times New Roman";
+color:#C00000'><o:p></o:p></span></p>
-<p><b>Week 1: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- take inventory, design constructs and primers, test competent
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-cells</b></p>
-<p>6/15/15 </p>
+<p class=MsoNormal align=center style='text-align:center'><i style='mso-bidi-font-style:
-<p>Worked on primer design quiz questions. Discussed shift of project direction with Justin based on recently published paper (Chen, 2015): <u><a href=3D"http://advances.sciencemag.org/content/1/5/e1500358.full" rel=3D"nofollow">http://advances.sciencemag.org/content/1/5/e1500358.full</a></u></p>
+normal'><span style='font-family:"Arial",sans-serif'>Courage is not the absence
-<p>Took inventory, created excel sheet online under <u>Protocols folder =E2=80=9CGenehackers 2015 Inventory=E2=80=9D</u></p>
+of fear, but rather the judgment that something is more important than fear.
-<p>Downloaded SnapGene Viewer to work on plasmid constructs. </p>
+The brave may not live forever, but the cautious do not live at all.<o:p></o:p></span></i></p>
-<p>6/16/15</p>
+<p class=MsoNormal align=center style='text-align:center'><i style='mso-bidi-font-style:
-<p>New idea for output system -create one construct with SasA/RpaA as activator of output molecule, another with LabA/RpaA as inhibitor (negative feedback loop) of output molecule. Based on article (Taniguchi, 2010). Started design on constructs, possibly 5 in total:</p>
+normal'><span style='font-family:"Arial",sans-serif'>--</span></i> <i
-<ol><li>Read-out activator (KaiCEE-RFP, RpaA, SasA) </li>
+style='mso-bidi-font-style:normal'><span style='font-family:"Arial",sans-serif'>Eduard
-<li>Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)</li>
+<span class=SpellE>Christoff</span> Philippe Gerard <span class=SpellE>Renaldi</span>,
-<li>Test gene (kaibc promoter, GFP)</li>
+Prince of <span class=SpellE>Genovia</span>, <span class=GramE>The</span>
-<li>Fusion SasA</li>
+Princess Diaries</span></i><i style='mso-bidi-font-style:normal'><span
-<li>Fusion KaiC-P </li></ol>
+style='font-size:16.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:
+"Times New Roman"'><o:p></o:p></span></i></p>
-<p>Last two based heavily on paper (Chen, 2015)</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'><o:p>&nbsp;</o:p></span></b></p>
-<p>Met with Jennifer Moran -need to do safety training before autoclaving liquids and other procedures, will accomplish once more people are back.</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 1: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>To Discuss: Is it worth having negative feedback regulator of RpaA/ output molecule?</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- take inventory, design
+constructs and primers, test competent cells</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>6/17/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Decided to use CikA as negative regulator/inhibitor instead of Lab A. CikA better characterized. Reviewed (Gutu. O=E2=80=99Shea, 2013). </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>6/18/15</p>
+<p class=MsoNormal align=center style='text-align:center'><span
-<p>Finished design on constructs 1, 2, 3 Deciding on RBS, perhaps need to use high efficiency promoters and lower efficiency RBS. Justin will email kaibc/Kai analog sequences. Decided on meeting 4pm Tuesday. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/15/15 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Worked on primer design quiz questions.
+Discussed shift of project direction with Justin based on recently published
+paper (Chen, 2015): </span><a
+href="http://advances.sciencemag.org/content/1/5/e1500358.full"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#1155CC'>http://advances.sciencemag.org/content/1/5/e1500358.full</span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>To Discuss: Specific RBS and promoter strengths on different genes. </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Took inventory, created excel sheet
+online under <u>Protocols folder “<span class=SpellE>Genehackers</span> 2015
+Inventory”</u></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Downloaded <span class=SpellE>SnapGene</span>
+Viewer to work on plasmid constructs. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Started Testing for Competent Cells</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
-<p>*Used Justin/Rust Lab protocol instead of iGEM/team protocol because did not have cmrR plates. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/16/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Labels:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<ul class=3D"small"><li>Plasmid of Interest- PJ006, containing KaiABC under kaiA, and kaibc promoters, Spec resistance, Conc=3D 100ng/uL</li></ul>
+mso-bidi-font-family:Arial;color:black'>New idea for output system -create one
-<li>Transformed plate- PJ006 MC 6/18/15 </li></ul>
+construct with <span class=SpellE>SasA</span>/<span class=SpellE>RpaA</span> as
-<p>Steps</p>
+activator of output molecule, another with <span class=SpellE>LabA</span>/<span
-<ol><li>Remove cells from freezer, incubate on ice </li>
+class=SpellE>RpaA</span> as inhibitor (negative feedback loop) of output
-<li>Add 1 uL DNA (100ng/uL) into competent cell tube </li>
+molecule. Based on article (Taniguchi, 2010). Started design on constructs,
-<li>Incubate tube on ice for 30 mins</li>
+possibly 5 in total:</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Incubate tube 42C water bath for 1 min heat shock</li>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Incubate tube ice 5 mins </li>
-<li>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</li></ol>
-<p>*Used LB from MJR Lab, will have to make LB tomorrow </p>
-<li>Incubate tube in shaker 37C for 1 hour</li>
-<li>Heat Spec plate in incubator as cold plate reduces efficiency,complete while cells shaking</li>
-<li>Collect pellet, spin 3000 rcf/gs for 3 mins </li>
-<li>Decant 800 uL of supernatant </li>
-<li>Use glass beads (5-6) per section (use sterile flame)</li>
-<li>Mix pellet, pipetted 200 uL in total, 180 to 0.90 section, 20 to 0.10 section </li>
-<li>Shake with beads and remove </li>
-<li>Incubate plate overnight 37C</li></ol>
-<p>6/19/15</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l16 level1 lfo1;
-<p><b>Checked on Transformed Plates</b></p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<p>Good growth on both sections. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
-<p>Transformation efficiency</p>
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
-<p><blockquote>0.10 Section</blockquote></p>
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-<p>      =3D (293 cfus) / ( ((1 uL x 100 ng/uL)/1000 uL soln)(20/200 uL plated))</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<p>		      =3D 293 cfus/0.01 ng DNA plated =3D <u>2.93 x 10^4 transformants/ng</u></p>
+color:black'>Read-out activator (<span class=SpellE>KaiCEE</span>-RFP, <span
+class=SpellE>RpaA</span>, <span class=SpellE>SasA</span>) <o:p></o:p></span></p>
-<p><b>To Discuss: How to improve Transformation Efficiency. </b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l16 level1 lfo1;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Read-out inhibitor (<span class=SpellE>KaiCEA</span>-RFP, <span
+class=SpellE>RpaA</span>, <span class=SpellE>CikA</span>)<o:p></o:p></span></p>
-<p><b>Made 500 mL LB Solution </b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l16 level1 lfo1;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Test gene (<span class=SpellE>kaibc</span> promoter, GFP)<o:p></o:p></span></p>
-<p><b>Made CM plates</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l16 level1 lfo1;
-<p>	</p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<p>Temperature of Freezer=3D 6 C, Chloramphenicol storage temperature=3D 2-8 C </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
-<p>125 uL of 50mg/ml cm used for every 250 uL plate soln made </p>
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
-<p>	Poured plates</p>
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-<p>	Need to label once plates set overnight</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Fusion <span class=SpellE>SasA</span><o:p></o:p></span></p>
-<p>Updated restock list, will go tomorrow to buy new supplies (check Inventory) </p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l16 level1 lfo1;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Fusion <span class=SpellE>KaiC</span>-P <o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>6/20/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Stock room closed :(, Will order things on Monday</p>
+mso-bidi-font-family:Arial;color:black'>Last two based heavily on paper (Chen,
-<p>Placed cmR plates in left cold room, bottom right corner of room behind some Rust plates </p>
+)</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Week 2: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- Finalize construct design, develop Competent Cells</b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>						6/22/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Worked on Week 1 Presentation</p>
+mso-bidi-font-family:Arial;color:black'>Met with Jennifer Moran -need to do
-<p>Streaked 1 Tube of Comp 2014 Cells on LB Only  Plate for Competent E.coli procedure -protocol under master list titled =E2=80=9CCompetent E.Coli 6/21/15=E2=80=9D</p>
+safety training before autoclaving liquids and other procedures, will
-<p>Started Mini-prep to extract Kai proteins. Inoculated 2 colonies, 1 each in 2mL LB + Spec medium. </p>
+accomplish once more people are back.</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>6/23/15 </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Cultured 5 colonies from LB only plate into 10 mL LB </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Week 1 meeting today</p>
-<p>Meeting Notes:</p>
-<p>=E2=86=92 Discussed project direction and construct design. Need to add terminator after RpaA. Need to decide whether or not fusion protein construct worth it. Given that RpaA might have some basal phosphorylation level, induced CikA should present some results. How the constructs are set up now, ideal tests can only be conducted with all three constructs. Don=E2=80=99t need to perhaps mutagenize cut sites as these plasmids will not be final bio-brick. Final biobrick would possibly only have CikA, SasA. Overall consensus is that CikA worth exploring. Need to develop primers ASAP. </p>
-<p>=E2=86=92 Need to develop more work on biosynthesis pathway and decide which molecule want to consider as well as what is focus of experiment. Want KaiABC as submitted biobrick so perhaps focusing on biosynthesis pathway is a bit ambitious. Need to consider perhaps alternative, simpler molecule. </p>
-<p>=E2=86=92 Will likely assemble using Gibson, order this free kit from RPI. </p>
-<p>Contact Danny for competent cells and Justin for Gibson primers</p>
-<p>6/24/15 </p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Worked on designing primers. Contacted Danny for competent cell procedure, will complete on Friday. Will conduct both CaCl2 and RbCl2 procedures. Already have streaked LB only plate for competent cells in cold room. </p>
+mso-bidi-font-family:Arial;color:#9900FF'>To Discuss: Is it worth having
+negative feedback regulator of <span class=SpellE>RpaA</span>/ output molecule?</span></i></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>6/25/15 </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Finished designing primers, will check with Kevin tomorrow. Inoculated two 5mL LB broths with 1-3 colonies each.  NEB Gibson Assembly Kit with competent cells arrived! </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>6/26/15 </p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/17/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Carried out competent cell procedure. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p> <u><a href=3D"http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf" rel=3D"nofollow">http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf</a></u></p>
+mso-bidi-font-family:Arial;color:black'>Decided to use <span class=SpellE>CikA</span>
+as negative regulator/inhibitor instead of Lab A. <span class=SpellE>CikA</span>
+better characterized. Reviewed (<span class=SpellE>Gutu</span>. O’Shea, 2013). </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>https://drive.google.com/open?id=3D0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV FIX THIS LINK</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Used both Rust Lab and RbCl2 as a comparison. </p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/18/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Week 3: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- Test efficiency of competent cells, start as much cloning as possible</b></p>
+mso-bidi-font-family:Arial;color:black'>Finished design on constructs 1, 2, 3
+Deciding on RBS, perhaps need to use high efficiency promoters and lower
+efficiency RBS. Justin will email <span class=SpellE>kaibc</span>/Kai analog
+sequences. Decided on meeting 4pm Tuesday. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>6/29/15</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>Conducted Transformation of CaCl2 and RbCl2. </p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<li>Remove cells from freezer, incubate tubes on ice </li>
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<li>Add 1 uL DNA (50 pg//uL) into competent cell tube </li>
-<li>Incubate tube on ice for 30 mins</li>
-<li>Incubate tube 42C water bath for 1 min heat shock</li>
-<li>Incubate tube ice 5 mins </li>
-<li>Rescue cells by pipetting 850 uL LB into tube (use sterile flame)</li>
-<li>Incubate tube in shaker 37C for 1 hour</li>
-<li>Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking</li>
-<li>Collect pellet, spin 3000 rcf/gs for 3 mins </li>
-<li>Decant 800 uL of supernatant </li>
-<li>Use glass beads (5-6) per section (use sterile flame)</li>
-<li>Mix pellet, pipetted 200 uL in total</li>
-<li>Shake with beads and remove </li>
-<li>Incubate plate overnight 37C</li></ol>
-<p>Spec on LB negative control culture overnight =3D-0.019 A (no growth at all)</p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#9900FF'>To Discuss: Specific RBS and promoter
+strengths on different genes. </span></i></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>=3D (52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 uL plated))</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>		      =3D 52 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>1.15 x 10^6 transformants/ng (Rust)</u></p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<p>=3D52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 uL plated))</p>
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
-<p>		      =3D 18 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>4.00 x 10^5 transformants/ng (RbCl2)</u></p>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
-<p>6/30/15</p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#4CBF26'>Started Testing for Competent Cells</span></u></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Primers for first constructs arrived, however at team meeting discussed how plasmids need to be re-designed to effectively compare SasA and CikA. Also CikA will need KaiB, and likely to add RpaB to be consistent with Chen et al. Constructs for Read-Out system were revamped, and constructs for Oscillation system were designed as well.</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>*Used Justin/Rust Lab protocol instead
+of <span class=SpellE>iGEM</span>/team protocol because did not have <span
+class=SpellE>cmrR</span> plates. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Week 4: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- Order final gBlocks and primers. Standardize and develop specific, in depth protocols. Practice Western Blots, start writing project report. </b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>7/20/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>gBlocks for Oscillation and Read-Out systems were modified. See Dropbox for final edits and modifications. </p>
+mso-bidi-font-family:Arial;color:black'>Labels:</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>7/21/15</p>
+<ul style='margin-top:0in' type=disc>
-<p>gBlocks for Oscillation and Read-Out systems were finally ordered. Primers were designed. </p>
+ <li class=MsoNormal style='color:black;mso-list:l15 level1 lfo2;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>Plasmid of
+     Interest- PJ006, containing <span class=SpellE>KaiABC</span> under <span
+     class=SpellE>kaiA</span>, and <span class=SpellE>kaibc</span> promoters,
+     Spec resistance, <span class=SpellE>Conc</span>= 100ng/<span class=SpellE>uL</span><o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l15 level1 lfo2;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>Transformed
+     plate- PJ006 MC 6/18/15 <o:p></o:p></span></li>
+</ul>
-<p>7/22/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Primers ordered. Reached out to grad advisers for Western Blotting techniques. Researched Gibson Assembly and Western Blot protocols. Will need to research GFP Protocols. </p>
+mso-bidi-font-family:Arial;color:black'>Steps</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>GFP Protocols</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p><a href=3D"http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf" rel=3D"nofollow">http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf</a></p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Remove cells from freezer, incubate on ice <o:p></o:p></span></p>
-<p>7/23</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p>Materials for practice western blot acquired.  Gibson Assembly protocol drafted. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 1 <span class=SpellE>uL</span> DNA (100ng/<span class=SpellE>uL</span>)
+into competent cell tube <o:p></o:p></span></p>
-<p>7/24</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p>Started western blot. See Rust Lab protocol. Slight changes include 7.5% gel used, cassette assembled not submerged in buffer. Primers diluted and placed in -20 fridge. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube on ice for 30 mins<o:p></o:p></span></p>
-<p>7/27</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p>Primary and secondary antibody staining accomplished. Experiments more clearly laid out. Need to start considering plan for pRha inducible promoter and how to alter stoichiometry.</p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube 42C water bath for 1 min heat shock<o:p></o:p></span></p>
-<p><b>Week 5: </b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p><b>Goals- Finalize and outline protocols, generate explanations for plasmids and background info for wiki and presentation, order materials, gblock assembly</b></p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube ice 5 mins <o:p></o:p></span></p>
-<p>7/28</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l3 level1 lfo3;
-<p>Western blot procedures expanded. See Aaron=E2=80=99s email about compatible backbones. This week discussed assays -will need to western blot for KaiA before investigating oscillatory system in order to characterize input L-Rhamnose to output Kai A production. Dilutions will occur on log scale first for L-Rhamnose. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>6.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Rescue cells by pipetting 900 <span class=SpellE>uL</span> LB into
+tube (use sterile flame)<o:p></o:p></span></p>
-<p>7/29</p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
-<p>Finished specific protocols -need to ask White lab for sonicator?. Looked up compatibility of backbones. pSB1,3,4 have pMB1 (copy number 100-300/cell), p15A (low-medium 10-12 copy), pSC101 (~5 copies/cell). Will need to construct primers for kaibc/GFP onto the SasA+CikA/SasA plasmids. </p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>*Used LB
+from MJR Lab, will have to make LB tomorrow </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>7/30/15</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo4;
-<p>Materials reviewed and listed. Meeting with Barry to talk about iGEM as a class. Went over protocols and methods. Allocated who is ordering what.</p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube in shaker 37C for 1 hour<o:p></o:p></span></p>
-<p>7/31/15</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo5;
-<p><b>Gibson Assembly</b></p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Heat Spec plate in incubator as cold
+plate reduces <span class=SpellE>efficiency,complete</span> while cells shaking<o:p></o:p></span></p>
-<p>5 uL of Gibson HiFi Master mix was used in each assembly reaction to minimize amount reagent used. Amount of blocks used, dependent on bps of each block relative to each other. Each gblock diluted in 20 uL of dH2O. Used standardized amount 50 ng of largest block in each assembly. Assembled on ice. Incubated on 50oC heatblock for 1 hour.</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo6;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Collect pellet, spin 3000 <span
+class=SpellE>rcf</span>/<span class=SpellE>gs</span> for 3 mins <o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo7;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decant 800 <span class=SpellE>uL</span>
+of supernatant <o:p></o:p></span></p>
-[[File:NotebookTable1.png]]
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo8;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use glass beads (5-6) per section (use
+sterile flame)<o:p></o:p></span></p>
-<html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo9;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Mix pellet, pipetted 200 <span
+class=SpellE>uL</span> in total, 180 to 0.90 section, 20 to 0.10 section <o:p></o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo10;
-<p>Assembled 5.5 times of 1X Master Mix (not on ice). Dilute 100 uM (100X) primers to 10X primers. 2 uL of primer and 18 uL of dH2O. Aliquoted 49 uL of Master Mix with 1 uL from Gibson Assembly Mix.</p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<p>Recipe Phusion Master Mix:</p>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
-<p>o   Phusion 5X GC Buffer =3D 10uL x 5.5 =3D 55 uL</p>
+Garamond;color:black'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p>o   dNTPs 10 mM  =3D 1 x 5.5 =3D5.5 uL</p>
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   F Primer MC003 10 uM =3D 2.5 x 5.5 =3D13.75 uL</p>
+mso-bidi-font-family:Arial;color:black'>Shake with beads and remove <o:p></o:p></span></p>
-<p>o   R Primer MC004 10 uM =3D 2.5 x 5.5=3D 13.75 uL</p>
-<p>o   Phusion DNAP =3D 0.5 x 5.5 =3D2.75 uL</p>
-<p>o   H2O =3D 32.5 x 178.75 uL</p>
-<p>o   DMSO =3D 1.5 x 5.5 =3D8.25 uL</p>
-<p>*Should have added only 170.5 uL (31 x 5.5)  =E2=80=93Mix slightly more dilute</p>
-<p>Thermocycler Settings: 30 cycles, 98o for 30s, 98o for 10s, 65o for 30s, 72o for 1 min, 72o 7 min, Hold 4o</p>
-<p><b>Week 6: </b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l2 level1 lfo11;
-<p><b>Goals- gblock assembly and Transformation</b></p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate plate overnight 37C<o:p></o:p></span></p>
-<p>8/3/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Decided on backbones:</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
-<p><b>            	</b>Oscillator MC001 =E2=80=93 Cmr (standard igem backbone for submitted biobrick)</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<p>Readout SasA MC002 =E2=80=93Amp (theoretically want to use with MC001 if successful) =C3=A0 need to add GFP + kai bc</p>
+color:black'>6/19/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>            	Readout SasA/CikA MC003 =E2=80=93Amp (=E2=80=9C  =E2=80=9C) =C3=A0 need to add GFP + kaibc</p>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p> 		KaiC Variants MC004-7- Cmr (would never use with MC001)</p>
-<p><b>Agarose Gel Casting</b></p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Made 50 mL of 1% Agarose gel. General procedure: added agarose and 1X TAE into ER flask, microwaved until boil, cool under water, poured into tray, added 0.75uL EtBr for visualizing, inserted combs, cool in cold room for 15-20 mins.</p>
+mso-bidi-font-family:Arial;color:#CC0000'>Checked on Transformed Plates</span></u></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Loaded 10uL of 6X loading dye into 50uL samples. Loaded 10 uL of 1 kB Plus DNA Ladder from Invitrogen. Seems to be issue w/amount of sample loaded =E2=80=93only 15-29 uL available. Run under 120V for 30 mins. Could be issue with evaporation in thermocycler. Gel too big for tray. Yields of products exist, however seems low. Issue with MC001 =E2=80=93no clear product visible. PCR should be redone.</p>
+mso-bidi-font-family:Arial;color:black'>Good growth on both sections. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation efficiency</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel6.png]]
+mso-bidi-font-family:Arial;color:black'>0.10 Section</span></i><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Extraction</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;=
+(293 <span class=SpellE>cfus</span>) / <span class=GramE>( (</span>(1 <span
+class=SpellE>uL</span> x 100 ng/<span class=SpellE>uL</span>)/1000 <span
+class=SpellE>uL</span> <span class=SpellE>soln</span>)(20/200 <span
+class=SpellE>uL</span> plated))</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Gel Weights:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>MC001 =E2=80=93N/A</p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:2'>                   </span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;=
-<p>MC004 -0.1536 g</p>
+<span class=SpellE>cfus</span>/0.01 ng DNA plated = <u>2.93 x 10^4 <span
-<p>MC005 -0.2048 g</p>
+class=SpellE>transformants</span>/ng</u></span><span style='font-size:16.0pt;
-<p>MC006 -0.0965 g</p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>MC007 -0.3962 g</p>
-<p> </p>
-<p>*1mg =3D 1uL</p>
-<p>Added 3x uL QG buffer to volume of gel and 1X uL of isopropanol to volume of gel.</p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>To redo PCR for MC001,4,5,6,7, 1X Master Mix for 15 reactions made. Two reactions for each construct.</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>PCR Master Mix Recipe-</p>
-<p>o   Phusion 5X GC Buffer =3D 150 uL</p>
-<p>o   dNTPs 10 mM  =3D 15  uL</p>
-<p>o   Phusion DNAP =3D 7.5 uL</p>
-<p>o   H2O =3D 465 uL</p>
-<p>o   DMSO =3D 22.5 uL</p>
-<p> </p>
-<p>Added 2.5uL of F and R Primers, 44 uL of Master Mix,  and 1uL of DNA for each sample. Thermocycler settings- 98o 2 min, 98o 15 s, 69o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>PCR Master mix also used for amplifying linear Cmr backbones (diluted in 10uL, used 1 uL of sample for Phusion PCR).</p>
-<p><b>8/4/15</b></p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis</b></p>
+mso-bidi-font-family:Arial;color:#9900FF'>To Discuss: How to improve
-<p>Made 100 mL of 1% Agarose gel. 10 uL, 1kB plus Ladder loaded. 35uL MC001, 20uL MC001, 34 uL MC004, 34 uL MC004, 33 uL of MC006,7,8 and Cmr backbones. Products from MC004,5,6,7 and Cmr Linearized backbones extracted using gel punches (borrowed from Rust Lab, need to order more to return). MC001 still not very good yield. Next step to purify MC004-7 and linearized backbones, redo Gibson and PCR of MC001 using gradient thermocycler.</p>
+Transformation Efficiency. </span></i></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel7.png]]
+mso-bidi-font-family:Arial;color:#4CBF26'>Made 500 mL LB Solution </span></u></b><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gibson Assembly</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>5 uL of Gibson HiFi Master mix, 1 uL PMC001_b1, 0.5965 uL PMC001_b2, 3.4305 uL H2O, heatblock for 1 hour 50oC.</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Master mix of 70 uL created (calculate ratio of 1X x 7/5)</p>
+mso-bidi-font-family:Arial;color:#4CBF26'>Made CM plates</span></u></b><span
-<p>o   Phusion 5X HC Buffer =3D 14 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>o   dNTPs 10 mM  =3D 1.4  uL</p>
-<p>o   Phusion DNAP =3D 0.7 uL</p>
-<p>o   H2O =3D 43.4 uL</p>
-<p>o   DMSO =3D 2.1 uL</p>
-<p>o   DNA =3D1.4 uL</p>
-<p>o   F Primer MC003=3D 3.5 uL</p>
-<p>o   R Primer MC004 =3D3.5 uL</p>
-<p>10uL Master mix aliquoted into 7 samples.</p>
-<p>Thermocycler settings- 98o 2 min, 98o 15 s, 60o, 62o, 64o, 66o, 68o, 70o, 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72.0o</p>
-<p>T=3D66.0o, G=3D6.0o for 30 cycles</p>
-<p><b>8/5/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gel Electrophoresis</p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span><span
-<p>Made 90 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye). Run for 120V, 30 mins. EtBr cloud on gel seen, only ladder shows visible bands. No other bands visible. Likely error with PCR and addition of EtBr.</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image:</b></p>
-</html>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
-[[File:UChicagoGel8.png]]
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Temperature
-<html>
+of Freezer= 6 C, Chloramphenicol storage temperature= 2-8 C </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Extraction</b></p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
-<p>Used Promega spin columns/buffer to concentrate in 15 uL of DNA </p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>125 <span
+class=SpellE>uL</span> of 50mg/ml cm used for every 250 <span class=SpellE>uL</span>
+plate <span class=SpellE>soln</span> made </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Purity Yields using nanodrop - C1 (Cam Backbone) -</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>C2 (Cam Backbone) -58.9 ng/uL</p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Poured
-<p>4 - 34.7</p>
+plates</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>4=E2=80=99 -</p>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>5 - 31.5</p>
-<p>6 - 36.3</p>
-<p>7 - 40.1</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b> </b></p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Need
-<p><b>PCR</b></p>
+to label once plates set overnight</span><span style='font-size:16.0pt;
-<p>To redo PCR for Gibson products of MC001, 75 uL of 1X PCR Master Mix</p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>PCR Master Mix Recipe-</p>
-<p>o   Phusion 5X HF Buffer =3D 15 uL</p>
-<p>o   dNTPs 10 mM  =3D 15  uL</p>
-<p>o   Phusion DNAP =3D .75 uL</p>
-<p>o   H2O =3D 46.5 uL</p>
-<p>o   DMSO =3D 2.25 uL</p>
-<p>o   DNA (products from Gibson 8/4)=3D 1.5uL</p>
-<p>o   F Primer MC003=3D 3.75uL</p>
-<p>o   R Primer MC004 =3D3.75uL</p>
-<p> </p>
-<p>10 uL of Master Mix aliquoted into each sample tube.</p>
-<p> </p>
-<p>To PCR PMC001_b1 for confirmation of block and analysis of primers</p>
-<p>50 uL of 1XPCR Master Mix Recipe-</p>
-<p>o   Phusion 5X HF Buffer =3D 10 uL</p>
-<p>o   dNTPs 10 mM  =3D 1  uL</p>
-<p>o   Phusion DNAP =3D .5 uL</p>
-<p>o   H2O=3D 31 uL</p>
-<p>o   DMSO =3D 1.5 uL</p>
-<p>o   DNA (pMC001_b1)=3D 1 uL</p>
-<p>o   F Primer MC005=3D 2.5uL</p>
-<p>o   R Primer MC006 =3D2.5uL</p>
-<p> </p>
-<p>Added 2.5uL of F and R Primers, 44 uL of Master Mix,  and 1uL of DNA for each sample..</p>
-<p>Thermocycler settings- 98o 2 min, 98o 15 s, 60o, 62o, 64o, 66o, 68o, 70o, 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72.0o</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>T=3D66.0o, G=3D6.0o for 30 cycles</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>8/6/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis</b></p>
+mso-bidi-font-family:Arial;color:black'>Updated restock list, will go tomorrow
-<p>Made 100 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye) and 1 50uL sample (divided into two wells, 42uL in one well 18 in the other). Run for 120V, 30 mins. No product clear enough to extract. Imaging gel shows faint products under 68,70, and 72o. Could mean issue with primers. Strangely, no product of right gBlock size seen. Again could be primers.</p>
+to buy new supplies (check Inventory) </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image:</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/20/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-[[File:UChicagoGel1.png]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Stock room closed :(, Will order things
+on Monday</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Placed <span class=SpellE>cmR</span>
+plates in left cold room, bottom right corner of room behind some Rust plates </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b> </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gibson Assembly:</b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Assembled purified biobricks MC004, MC005, MC006, MC007 into Cam backbone. Used following recipe based on bps of insert and backbone. </p>
-</html>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 2: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-[[File:UChicagoTable2.png]]
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- Finalize construct design,
+develop Competent Cells</span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Transformation of Assembled products:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>DNA straight from Gibson Assembly Reaction was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:6'>                                                          </span>6/22/15</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<ol><li>Remove cells from freezer, incubate tubes on ice </li>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Add 1 uL DNA (50 pg//uL) into competent cell tube </li>
+mso-bidi-font-family:Arial;color:black'>Worked on Week 1 Presentation</span><span
-<li>Incubate tube on ice for 30 mins</li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Incubate tube 42C water bath for 1 min heat shock</li>
-<li>Incubate tube ice 5 mins </li>
-<li>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</li>
-<li>Incubate tube in shaker at 37C/1100 RPM for 1 hour</li>
-<li>Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking</li>
-<li>Collect pellet, spin 3000 rcf/gs for 3 mins </li>
-<li>Decant 800 uL of supernatant </li>
-<li>Use glass beads (5-6) per section (use sterile flame)</li>
-<li>Mix pellet, pipetted 200 uL in total</li>
-<li>Shake with beads and remove </li>
-<li>Incubate plate overnight 37C</li></ol>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Streaked 1 Tube of Comp 2014 Cells on
+LB Only &nbsp;Plate for Competent E.coli procedure -protocol under master list
+titled “Competent <span class=SpellE>E.Coli</span> 6/21/15”</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>*Negative control w/no transformed DNA resulted in 0 colonies. Negative control set up on 8/7.</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Started Mini-prep to extract Kai
+proteins. Inoculated 2 colonies, 1 each in 2mL LB + Spec medium. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoPlate3.png]]
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<html>
-<p><b>PCR</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
-<p>PCR of 8/3 Gibson PCR, 8/4 Gibson PCR conducted for further amplification. Block 4.1 PCR as positive control. Block 1.1 and 1.2 PCR run to increase DNA amount in hopes of Gibson from amplified blocks. 50 uL of sample for each PCR (5 samples in total). </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/23/15 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Added Ingredients to individual Samples</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   Phusion 5X HC Buffer =3D 10 uL</p>
+mso-bidi-font-family:Arial;color:black'>Cultured 5 colonies from LB only plate
-<p>o   dNTPs 10 mM  =3D 1 uL</p>
+into 10 mL LB </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   Phusion DNAP =3D 0.5 uL</p>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>o   H2O =3D 31.0 uL</p>
-<p>o   DMSO =3D 1.5 uL</p>
-<p>o   DNA =3D1.0 uL</p>
-<p>o   F Primer =3D 2.5 uL</p>
-<p>o   R Primer =3D2.5 uL</p>
-<p>10uL Master mix aliquoted into 7 samples.</p>
-<p>Thermocycler settings- 98o 2 min, 98o 15 s, 70o, 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>Primers for each sample</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gibson 8/3 -MC003/MC004</p>
+mso-bidi-font-family:Arial;color:black'>Week 1 meeting today</span><span
-<p>Gibson 8/4 -MC003/MC004</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>pMC001_b1 -MC005/MC006</p>
-<p>pMC001_b2 -MC007/MC008</p>
-<p>pMC004_b1- MC019/MC020</p>
-<p>*Upon further examination, should have used MC003 for pMC004_b1</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Meeting Notes:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/7/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis</b></p>
+mso-bidi-font-family:"Times New Roman";color:black'>&#8594;</span><span
-<p>1% Agarose gel run of PCR products from 8/6. Could not see products under blue light. Under UV light, products seemed more specific. Still not as strong as in previous gels. Perhaps need to troubleshoot PCR better. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> Discussed project direction and construct design. Need to add
+terminator after <span class=SpellE>RpaA</span>. Need to decide whether or not
+fusion protein construct worth it. Given that <span class=SpellE>RpaA</span>
+might have some basal phosphorylation level, induced <span class=SpellE>CikA</span>
+should present some results. How the constructs are set up now, ideal tests can
+only be conducted with all three constructs. Don’t need to perhaps mutagenize
+cut sites as these plasmids will not be final bio-brick. Final <span
+class=SpellE>biobrick</span> would possibly only have <span class=SpellE>CikA</span>,
+<span class=SpellE>SasA</span>. Overall consensus is that <span class=SpellE>CikA</span>
+worth exploring. Need to develop primers ASAP. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman";color:black'>&#8594;</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> Need to develop more work on biosynthesis pathway and decide
+which molecule want to consider as well as what is focus of experiment. Want <span
+class=SpellE>KaiABC</span> as submitted <span class=SpellE>biobrick</span> so
+perhaps focusing on biosynthesis pathway is a bit ambitious. Need to consider
+perhaps alternative, simpler molecule. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel9.png]]
+mso-bidi-font-family:"Times New Roman";color:black'>&#8594;</span><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> <span class=GramE>Will</span> likely assemble using Gibson, order
+this free kit from RPI. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Transformation Results</b> </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Used iPhone application =E2=80=9CColonyCount=E2=80=9D to assist in counting plates.</p>
+mso-bidi-font-family:Arial;color:black'>Contact Danny for competent cells and
-<p>Plate Numbers are indicated on lower left of the pictures.</p>
+Justin for Gibson primers</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Average is 321 colonies.</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/24/15 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-[[File:UChicagoColonies1_1.jpg]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoColonies1_2.jpg]]
+mso-bidi-font-family:Arial;color:black'>Worked on designing primers. Contacted
-[[File:UChicagoColonies1_3.jpg]]
+Danny for competent cell procedure, will complete on Friday. Will conduct both
-[[File:UChicagoColonies1_4.jpg]]
+CaCl2 and RbCl2 procedures. Already have streaked LB only plate for competent
+cells in cold room. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Transformation Efficiency </b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/25/15 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Used 1ul of 50pg/ul  of DNA </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Finished designing primers, will check
+with Kevin tomorrow. Inoculated two 5mL LB broths with 1-3 colonies each.
+&nbsp;NEB Gibson Assembly Kit with competent cells arrived! </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>4: 287 / 5*10^-5 =3D 5.74*10^6 cfu/ug</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>5: 328 / 5*10^-5 =3D 6.56*10^6 cfu/ug</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>6: 414 / 5*10^-5 =3D 8.28*10^6 cfu/ug</p>
-<p>7: 254 / 5*10^-5 =3D 5.08*10^6 cfu/ug</p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/26/15 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Primers</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Designed Sequencing primers as well as new primers for pMC001. Primers made specifically for oscillator plasmid -overhangs incorporated to make primers longer and more specific. </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Prepared PCR of products seen on gel in morning (G1, G2, 001b1, 001b2, 004b1, used 1 uL of leftover PCR reaction). Used Q5 High Fidelity polymerase, as no Phusion available. </p>
+mso-bidi-font-family:Arial;color:black'>Carried out competent cell procedure. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Ingredients:</p>
+<p class=MsoNormal><a
-<p>	Q5 High-Fidelity 2X Master Mix- 25 uL</p>
+href="http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf"><span
-<p>	DNA -1 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<p>	F Primer -2.5 uL</p>
+color:#1155CC;background:white'>http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf</span></a><span
-<p>	R Primer -2.5 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	H2O -19 uL </p>
-<p>Thermocycler Settings: 98C 30s, 98C 10s, 65C for 30s, 72C for 30s, 72C for 2mins, hold at 4C</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>30 cycles </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Prepared Colony PCR To confirm inserts of pMC004,5,6,7. Used Taq DNA polymerase instead of phusion. </p>
+<p class=MsoNormal><a
+href="https://drive.google.com/open?id=0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#1155CC'>https://drive.google.com/open?id=0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV</span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<ol><li>Pick single colony from plate, place in 50uL of dH2O (acts as DNA template)</li>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Add following PCR 1X Master Mix for Taq:</li></ol>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>5 uL 10X buffer</p>
-<p>1 uL dNTPs</p>
-<p>1 uL 10 uM primer stock-VF2 and VR</p>
-<p>1 uL DNA stock</p>
-<p>0.5 uL Taq</p>
-<p>41.5 uL H2O </p>
-<p>Thermocycler Settings: 95C 2mins, 95C 15s, 55C 15s, 68C 45s (30 cycles), 68C 10m, hold 4C</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Used both Rust Lab and RbCl2 as a
+comparison. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>-> Extend to 1min per kb (look up on product sheet)</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>8/9/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 3: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Ran 1% Agarose gel 120V, 30 mins. Ran both Colony PCR and Q5 PCR. 5uL each sample loaded. Different ladder used (Quick Load Purple 2-Log from NEB. Same amount of EtBr (0.75 uL) used. </p>
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- Test efficiency of competent
+cells, start as much cloning as possible</span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel10.png]]
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<html>
-<p><b>8/10/15</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/29/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gel repeated, this time using leftover 45uL of sample. 1kB Plus invitrogen ladder used. </p>
+mso-bidi-font-family:Arial;color:black'>Conducted Transformation of CaCl2 and
+RbCl2. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo12;
-[[File:UChicagoGel11.png]]
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Remove cells from freezer, incubate
+tubes on ice <o:p></o:p></span></p>
-<p><b>Innoculation-</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo13;
-<p>Due to failure of Colony PCR, colonies were inoculated and incubated. Will conduct direct miniprep on 8/11 and sequence to sequence. This should help in determining if there is a problem with primers/insert or with the PCR. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 1 <span class=SpellE>uL</span> DNA
+(50 <span class=SpellE>pg</span>//<span class=SpellE>uL</span>) into competent
+cell tube <o:p></o:p></span></p>
-<p><b>Gibson Assembly-</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo14;
-<p>Gibson assembly of BH001_b1 and Cam backbone conducted. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube on ice for 30 mins<o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo15;
-[[File:UChicagoTable5.png]]
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube 42C water bath for 1 min
+heat shock<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo16;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>19.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube ice 5 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo17;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>20.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Rescue cells by pipetting 850 <span
+class=SpellE>uL</span> LB into tube (use sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo18;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>21.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube in shaker 37C for 1 hour<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo19;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>22.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Heat Cam plate in incubator as cold
+plate reduces <span class=SpellE>efficiency,complete</span> while cells shaking<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo20;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>23.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Collect pellet, spin 3000 <span
+class=SpellE>rcf</span>/<span class=SpellE>gs</span> for 3 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo21;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>24.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decant 800 <span class=SpellE>uL</span>
+of supernatant <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo22;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>25.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use glass beads (5-6) per section (use
+sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo23;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>26.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Mix pellet, pipetted 200 <span
+class=SpellE>uL</span> in total<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo24;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>27.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Shake with beads and remove <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l11 level1 lfo25;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>28.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate plate overnight 37C<o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Spec on LB negative control culture
+overnight =-0.019 A (no growth at all)</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>= (52 <span
+class=SpellE>cfus</span>) / <span class=GramE>( (</span>(1 <span class=SpellE>uL</span>
+x 50 <span class=SpellE>pg</span>/<span class=SpellE>uL</span> x
+ng/1000pg)/1000uL <span class=SpellE>soln</span>))*((180/200 <span
+class=SpellE>uL</span> plated))</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:2'>                   </span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;=
+<span class=SpellE>cfus</span><span class=GramE>/(</span>4.5 x 10^-5) ng DNA
+plated =<u>1.15 x 10^6 <span class=SpellE>transformants</span>/ng (Rust)</u></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>=52 <span
+class=SpellE>cfus</span>) / <span class=GramE>( (</span>(1 <span class=SpellE>uL</span>
+x 50 <span class=SpellE>pg</span>/<span class=SpellE>uL</span> x 1ng/1000pg)/1000uL
+<span class=SpellE>soln</span>))*((180/200 <span class=SpellE>uL</span>
+plated))</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:2'>                   </span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;=
+<span class=SpellE>cfus</span><span class=GramE>/(</span>4.5 x 10^-5) ng DNA
+plated =<u>4.00 x 10^5 <span class=SpellE>transformants</span>/ng (RbCl2)</u></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>6/30/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers for first constructs arrived,
+however at team meeting discussed how plasmids need to be re-designed to effectively
+compare <span class=SpellE>SasA</span> and <span class=SpellE>CikA</span>. Also
+<span class=SpellE>CikA</span> will need <span class=SpellE>KaiB</span>, and
+likely to add <span class=SpellE>RpaB</span> to be consistent with Chen et al.
+Constructs for Read-Out system were revamped, and constructs for Oscillation
+system were designed as well.</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 4: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- Order final <span
+class=SpellE>gBlocks</span> and primers. Standardize and develop specific, in
+depth protocols. Practice Western Blots, start writing project report. </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>7/20/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span class=GramE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>gBlocks</span></span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> for Oscillation and Read-Out systems were modified. See Dropbox
+for final edits and modifications. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>7/21/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span class=GramE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>gBlocks</span></span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> for Oscillation and Read-Out systems were finally ordered.
+Primers were designed. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>7/22/15</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers ordered. Reached out to grad
+advisers for Western Blotting techniques. Researched Gibson Assembly and
+Western Blot protocols. Will need to research GFP Protocols. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>GFP Protocols</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><a
+href="http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#1155CC'>http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf</span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/23</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Materials for practice western blot
+acquired. &nbsp;Gibson Assembly protocol drafted. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/24</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Started western blot. See Rust Lab
+protocol. Slight changes include 7.5% gel used, cassette assembled not
+submerged in buffer. Primers diluted and placed in -20 fridge. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/27</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primary and secondary antibody staining
+accomplished. Experiments more clearly laid out. Need to start considering plan
+for <span class=SpellE>pRha</span> inducible promoter and how to alter
+stoichiometry.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 5: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- Finalize and outline
+protocols, generate explanations for plasmids and background info for wiki and
+presentation, order materials, <span class=SpellE>gblock</span> assembly</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/28</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Western blot procedures expanded. See
+Aaron’s email about compatible backbones. This week discussed assays -will need
+to western blot for <span class=SpellE>KaiA</span> before investigating
+oscillatory system in order to characterize input L-<span class=SpellE>Rhamnose</span>
+to output Kai A production. Dilutions will occur on log scale first for L-<span
+class=SpellE>Rhamnose</span>. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/29</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Finished specific protocols -need to
+ask White lab for <span class=SpellE>sonicator</span><span class=GramE>?.</span>
+Looked up compatibility of backbones. pSB1<span class=GramE>,3,4</span> have
+pMB1 (copy number 100-300/cell), p15A (low-medium 10-12 copy), pSC101 (~5
+copies/cell). Will need to construct primers for <span class=SpellE>kaibc</span>/GFP
+onto the <span class=SpellE>SasA+CikA</span>/<span class=SpellE>SasA</span>
+plasmids. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/30/15</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Materials reviewed and listed. Meeting
+with Barry to talk about <span class=SpellE>iGEM</span> as a class. Went over
+protocols and methods. Allocated who is ordering what.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7/31/15</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>5 <span class=SpellE>uL</span> of
+Gibson <span class=SpellE>HiFi</span> Master mix was used in each assembly
+reaction to minimize amount reagent used. Amount of blocks used, dependent on
+bps of each block relative to each other. Each <span class=SpellE>gblock</span>
+diluted in 20 <span class=SpellE>uL</span> of dH<sub>2</sub>O. Used
+standardized amount 50 ng of largest block in each assembly. Assembled on ice.
+Incubated on 50<sup>o</sup>C <span class=SpellE>heatblock</span> for 1 hour.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><!--[if gte vml 1]><v:shapetype id="_x0000_t75" coordsize="21600,21600"
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+ stroked="f">
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+ <v:imagedata src="..//wiki/images/4/46/UChicago2015Notebook_filesimage001.png" o:title="mage 1"/>
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+src="..//wiki/images/a/ab/UChicago2015Notebook_filesimage002.jpg" align=left hspace=12 alt="mage 1.png" v:shapes="Picture_x0020_1"><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Assembled 5.5 times of 1X Master Mix
+(not on ice). Dilute 100 <span class=SpellE>uM</span> (100X) primers to 10X
+primers. 2 <span class=SpellE>uL</span> of primer and 18 <span class=SpellE>uL</span>
+of dH<sub>2</sub>O. Aliquoted 49 <span class=SpellE>uL</span> of Master Mix
+with 1 <span class=SpellE>uL</span> from Gibson Assembly Mix.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Recipe <span class=SpellE>Phusion</span>
+Master Mix:</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X GC Buffer = 10uL x 5.5 = 55 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 1 x 5.5 =5.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC003 10 <span
+class=SpellE>uM</span> = 2.5 x 5.5 =13.75 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC004 10 <span
+class=SpellE>uM</span> = 2.5 x 5.5= 13.75 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 0.5 x 5.5 =2.75 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 32.5
+x 178.75 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 1.5 x 5.5 =8.25
+<span class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>*Should have added only 170.5 <span
+class=SpellE>uL</span> (31 x 5.5<span class=GramE>) &nbsp;–</span>Mix slightly
+more dilute</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings: 30 cycles, 98<sup>o</sup>
+for 30s, 98<sup>o</sup> for 10s, 65<sup>o</sup> for 30s, 72<sup>o</sup> for 1
+min, 72<sup>o</sup> 7 min, Hold 4<sup>o</sup></span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Week 6: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#1155CC'>Goals- <span class=SpellE>gblock</span>
+assembly and Transformation</span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/3/15</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decided on backbones:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span
+style='mso-tab-count:1'>        </span></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Oscillator
+MC001 – <span class=SpellE>Cmr</span> (standard <span class=SpellE>igem</span>
+backbone for submitted <span class=SpellE>biobrick</span>)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Readout <span
+class=SpellE>SasA</span> MC002 –Amp (theoretically want to use with MC001 if
+successful) à need to add GFP + kai <span class=SpellE><span class=GramE>bc</span></span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span
+style='mso-tab-count:1'>        </span>Readout <span class=SpellE>SasA</span>/<span
+class=SpellE>CikA</span> MC003 –Amp (<span class=GramE>“ &nbsp;“</span>) à need
+to add GFP + <span class=SpellE>kaibc</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:2'>                   </span><span
+class=SpellE>KaiC</span> Variants MC004-7- <span class=SpellE>Cmr</span> (would
+never use with MC001)</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Agarose Gel Casting</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 50 mL of 1% Agarose gel. General
+procedure: added agarose and 1X TAE into ER flask, microwaved until boil, cool
+under water, poured into tray, added 0.75uL EtBr for visualizing, inserted
+combs, cool in cold room for 15-20 mins.</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Loaded 10uL of 6X loading dye into 50uL
+samples. Loaded 10 <span class=SpellE>uL</span> of 1 kB Plus DNA Ladder from
+Invitrogen. Seems to be issue w/amount of sample loaded –only 15-29 <span
+class=SpellE>uL</span> available. Run under 120V for 30 mins. Could be issue
+with evaporation in thermocycler. Gel too big for tray. Yields of products
+exist, however seems low. Issue with MC001 –no clear product visible. PCR
+should be redone.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
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+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Extraction</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Weights:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC001 –N/A</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC004 -0.1536 g</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC005 -0.2048 g</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC006 -0.0965 g</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC007 -0.3962 g</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>*1mg = 1uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Added 3x <span class=SpellE>uL</span>
+QG buffer to volume of gel and 1X <span class=SpellE>uL</span> of isopropanol
+to volume of gel.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>To redo PCR for MC001<span class=GramE>,4,5,6,7</span>,
+X Master Mix for 15 reactions made. Two reactions for each construct.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Master Mix Recipe-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X GC Buffer = 150 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 15 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 7.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 465 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 22.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Added 2.5uL of F and R Primers, 44 <span
+class=SpellE>uL</span> of Master Mix<span class=GramE>, &nbsp;and</span> 1uL of
+DNA for each sample. Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup>
+s, 69<sup>o</sup> 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10 min, 4<sup>o</sup>
+hold.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Master <span class=GramE>mix</span>
+also used for amplifying linear <span class=SpellE>Cmr</span> backbones
+(diluted in 10uL, used 1 <span class=SpellE>uL</span> of sample for <span
+class=SpellE>Phusion</span> PCR).</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/4/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 100 mL of 1% Agarose gel. 10 <span
+class=SpellE>uL</span>, 1kB plus Ladder loaded. 35uL MC001, 20uL MC001, 34 <span
+class=SpellE>uL</span> MC004, 34 <span class=SpellE>uL</span> MC004, 33 <span
+class=SpellE>uL</span> of MC006<span class=GramE>,7,8</span> and <span
+class=SpellE>Cmr</span> backbones. Products from MC004<span class=GramE>,5,6,7</span>
+and <span class=SpellE>Cmr</span> Linearized backbones extracted using gel
+punches (borrowed from Rust Lab, need to order more to return). MC001 still not
+very good yield. Next step to purify MC004-7 and linearized backbones, redo
+Gibson and PCR of MC001 using gradient thermocycler.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_3" o:spid="_x0000_i1063"
+ type="#_x0000_t75" alt="https://lh4.googleusercontent.com/vhr9tI0AP8GjCG1NHKQ1TBox7RaB6XEsB5IFctg6cGxmBqjaYWc2jYikx9hAZOvmlMtz2s1ksbRWM0hC6OUiM2y2kx-8zU3SFT_Edk-ZqvzjOpMiR0sHZ0xY_Y75RXMdAiL2nrg"
+ style='width:705pt;height:672.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/2/23/UChicago2015Notebook_filesimage005.png" o:title="vhr9tI0AP8GjCG1NHKQ1TBox7RaB6XEsB5IFctg6cGxmBqjaYWc2jYikx9hAZOvmlMtz2s1ksbRWM0hC6OUiM2y2kx-8zU3SFT_Edk-ZqvzjOpMiR0sHZ0xY_Y75RXMdAiL2nrg"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=940 height=897
+src="..//wiki/images/7/74/UChicago2015Notebook_filesimage006.png"
+alt="https://lh4.googleusercontent.com/vhr9tI0AP8GjCG1NHKQ1TBox7RaB6XEsB5IFctg6cGxmBqjaYWc2jYikx9hAZOvmlMtz2s1ksbRWM0hC6OUiM2y2kx-8zU3SFT_Edk-ZqvzjOpMiR0sHZ0xY_Y75RXMdAiL2nrg"
+v:shapes="Picture_x0020_3"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>5 <span class=SpellE>uL</span> of
+Gibson <span class=SpellE>HiFi</span> Master mix, 1 <span class=SpellE>uL</span>
+PMC001_b1, 0.5965 <span class=SpellE>uL</span> PMC001_b2, 3.4305 <span
+class=SpellE>uL</span> H<sub>2</sub>O, <span class=SpellE>heatblock</span> for
+hour 50<sup>o</sup>C.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Master <span class=GramE>mix</span> of
+<span class=SpellE>uL</span> created (calculate ratio of 1X x 7/5)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HC Buffer = 14 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 1.4 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 0.7 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 43.4 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 2.1 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA =1.4 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC003= 3.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC004 =3.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>10uL Master mix aliquoted into 7 samples.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98<sup>o</sup> 2
+min, 98<sup>o</sup> 15 s, 60<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>,
+<sup>o</sup>, 68<sup>o</sup>, 70<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup>
+min, 72<sup>o</sup> 10 min, 4<sup>o</sup> hold.</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>,
+.0<sup>o</sup>, 63.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>,
+.0<sup>o</sup></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup>
+for 30 cycles</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/5/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 90 mL of 1% Agarose gel. Loaded
+uL of 7 <span class=GramE>samples(</span>+loading dye). Run for 120V, 30
+mins. EtBr cloud on gel seen, only ladder shows visible bands. No other bands
+visible. Likely error with PCR and addition of EtBr.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_4"
+ o:spid="_x0000_i1062" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/ep3k5EhBnMexsc-fQ-22FFqWJRGb3vGSet4vO6KpuOEVcFUzp2DGnVNpFkpcQpiX10kcAqz_7IgsH0qTP76UXscOgzGidyp2zjqUFnsAbz5Ql6aApcrL0bBNjreWJP6HAfeNEwg"
+ style='width:760.5pt;height:616.5pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/4a/UChicago2015Notebook_filesimage007.png" o:title="ep3k5EhBnMexsc-fQ-22FFqWJRGb3vGSet4vO6KpuOEVcFUzp2DGnVNpFkpcQpiX10kcAqz_7IgsH0qTP76UXscOgzGidyp2zjqUFnsAbz5Ql6aApcrL0bBNjreWJP6HAfeNEwg"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1014 height=822
+src="..//wiki/images/6/62/UChicago2015Notebook_filesimage008.png"
+alt="https://lh5.googleusercontent.com/ep3k5EhBnMexsc-fQ-22FFqWJRGb3vGSet4vO6KpuOEVcFUzp2DGnVNpFkpcQpiX10kcAqz_7IgsH0qTP76UXscOgzGidyp2zjqUFnsAbz5Ql6aApcrL0bBNjreWJP6HAfeNEwg"
+v:shapes="Picture_x0020_4"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Extraction</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Used <span class=SpellE>Promega</span>
+spin columns/buffer to concentrate in 15 <span class=SpellE>uL</span> of DNA </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;background:lime'>Purity Yields using <span
+class=SpellE>nanodrop</span> - C1 (Cam Backbone) -</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>C2 (Cam Backbone) -58.9 ng/<span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>4 - 34.7</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>4’ -</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>5 - 31.5</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>6 - 36.3</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;background:lime'>7 - 40.1</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>To redo PCR for Gibson products of
+MC001, 75 <span class=SpellE>uL</span> of 1X PCR Master Mix</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Master Mix Recipe-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 15 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 15 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = .75 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 46.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 2.25 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA (products from
+Gibson 8/4)= 1.5uL</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC003= 3.75uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC004 =3.75uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>10 <span class=SpellE>uL</span> of
+Master Mix aliquoted into each sample tube.</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>To PCR PMC001_b1 for confirmation of
+block and analysis of primers</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>50 <span class=SpellE>uL</span> of
+XPCR Master Mix Recipe-</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 10 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 1 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = .5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O= 31 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 1.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA (pMC001_b1)= 1 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC005= 2.5uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC006 =2.5uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Added 2.5uL of F and R Primers, 44 <span
+class=SpellE>uL</span> of Master Mix<span class=GramE>, &nbsp;and</span> 1uL of
+DNA for each sample..</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98<sup>o</sup> 2
+min, 98<sup>o</sup> 15 s, 60<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>,
+<sup>o</sup>, 68<sup>o</sup>, 70<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup>
+min, 72<sup>o</sup> 10 min, 4<sup>o</sup> hold.</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>,
+.0<sup>o</sup>, 63.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>,
+.0<sup>o</sup></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup>
+for 30 cycles</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/6/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 100 mL of 1% Agarose gel. Loaded
+uL of 7 <span class=GramE>samples(</span>+loading dye) and 1 50uL sample
+(divided into two wells, 42uL in one well 18 in the other). Run for 120V, 30
+mins. No product clear enough to extract. Imaging gel shows faint products
+under 68<span class=GramE>,70</span>, and 72<sup>o</sup>. Could mean issue with
+primers. Strangely, no product of right <span class=SpellE>gBlock</span> size
+seen. Again could be primers.</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_5"
+ o:spid="_x0000_i1061" type="#_x0000_t75" alt="igure 4.png" style='width:471.75pt;
+ height:381.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/2/2f/UChicago2015Notebook_filesimage009.png" o:title="igure 4"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=629 height=509
+src="..//wiki/images/f/f2/UChicago2015Notebook_filesimage010.jpg" alt="igure 4.png" v:shapes="Picture_x0020_5"><![endif]></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Assembled purified <span class=SpellE>biobricks</span>
+MC004, MC005, MC006, <span class=GramE>MC007</span> into <span class=SpellE>Cam</span>
+backbone. Used following recipe based on bps of insert and backbone. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_6"
+ o:spid="_x0000_i1060" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/zyijYOnOqRFCBDdEEMBCRewGmCzJVhakw5hLpdUOsgGPeQARFugu30Vc4sBXfhFJ-WA0uuAX13-2qz6DuMIy-dsK0BJTfKWl1SanXnyWPnYCab_E-wUmAR3z0oDKEkclhEaFrM0"
+ style='width:895.5pt;height:120.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/a/a5/UChicago2015Notebook_filesimage011.png" o:title="zyijYOnOqRFCBDdEEMBCRewGmCzJVhakw5hLpdUOsgGPeQARFugu30Vc4sBXfhFJ-WA0uuAX13-2qz6DuMIy-dsK0BJTfKWl1SanXnyWPnYCab_E-wUmAR3z0oDKEkclhEaFrM0"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1194 height=161
+src="..//wiki/images/7/78/UChicago2015Notebook_filesimage012.png"
+alt="https://lh3.googleusercontent.com/zyijYOnOqRFCBDdEEMBCRewGmCzJVhakw5hLpdUOsgGPeQARFugu30Vc4sBXfhFJ-WA0uuAX13-2qz6DuMIy-dsK0BJTfKWl1SanXnyWPnYCab_E-wUmAR3z0oDKEkclhEaFrM0"
+v:shapes="Picture_x0020_6"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation of Assembled products:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>DNA straight from Gibson Assembly
+Reaction was transformed into competent cells. RbCl2 competent cells used.
+Efficiency of cells: <u>4.00 x 10^5 <span class=SpellE>transformants</span>/ng
+(RbCl2) </u></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Remove cells from freezer, incubate tubes on ice <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 1 <span class=SpellE>uL</span> DNA (50 <span class=SpellE>pg</span>//<span
+class=SpellE>uL</span>) into competent cell tube <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube on ice for 30 mins<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube 42C water bath for 1 min heat shock<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube ice 5 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>6.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Rescue cells by pipetting 900 <span class=SpellE>uL</span> LB into
+tube (use sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>7.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate tube in shaker at 37C/1100 RPM for 1 hour<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>8.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Heat Cam plate in incubator as cold plate reduces <span
+class=SpellE>efficiency,complete</span> while cells shaking<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>9.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Collect pellet, spin 3000 <span class=SpellE>rcf</span>/<span
+class=SpellE>gs</span> for 3 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>10.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decant 800 <span class=SpellE>uL</span>
+of supernatant <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>11.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use glass beads (5-6) per section (use
+sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>12.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Mix pellet, pipetted 200 <span
+class=SpellE>uL</span> in total<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>13.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Shake with beads and remove <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l5 level1 lfo26;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>14.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate plate overnight 37C<o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_7"
+ o:spid="_x0000_i1059" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/s_QBM9SKfAUn9wjfvTWl9K00NTt1kxDu5VMMaLhIMAGa_yQ1-Wauhe7DvycjA8ELKmiv1cnttv3XJOhb_79JO_NisWwYJr9KmP_U6VxF8cb5wChdZvi1yCBwYSUFd84SuQP0jSE"
+ style='width:1488.75pt;height:1413pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/9/99/UChicago2015Notebook_filesimage013.png" o:title="s_QBM9SKfAUn9wjfvTWl9K00NTt1kxDu5VMMaLhIMAGa_yQ1-Wauhe7DvycjA8ELKmiv1cnttv3XJOhb_79JO_NisWwYJr9KmP_U6VxF8cb5wChdZvi1yCBwYSUFd84SuQP0jSE"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1985 height=1884
+src="..//wiki/images/e/e9/UChicago2015Notebook_filesimage014.png"
+alt="https://lh3.googleusercontent.com/s_QBM9SKfAUn9wjfvTWl9K00NTt1kxDu5VMMaLhIMAGa_yQ1-Wauhe7DvycjA8ELKmiv1cnttv3XJOhb_79JO_NisWwYJr9KmP_U6VxF8cb5wChdZvi1yCBwYSUFd84SuQP0jSE"
+v:shapes="Picture_x0020_7"><![endif]></span><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>*Negative control w/no transformed DNA
+resulted in 0 colonies. Negative control set up on 8/7.</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR of 8/3 Gibson PCR, 8/4 Gibson PCR
+conducted for further amplification. Block 4.1 PCR as positive control. Block
+.1 and 1.2 PCR run to increase DNA amount in hopes of Gibson from amplified
+blocks. 50 <span class=SpellE>uL</span> of sample for each PCR (5 samples in
+total). </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Added Ingredients to individual Samples</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HC Buffer = 10 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 1 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 0.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 31.0 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 1.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA =1.0 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer = 2.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer =2.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>10uL Master mix aliquoted into 7 samples.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98<sup>o</sup> 2
+min, 98<sup>o</sup> 15 s, 70<sup>o</sup>, 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup>
+min, 4<sup>o</sup> hold.</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers for each sample</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson 8/3 -MC003/MC004</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson 8/4 -MC003/MC004</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>pMC001_b1 -MC005/MC006</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>pMC001_b2 -MC007/MC008</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>pMC004_b1-</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> MC019/MC020</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>*Upon further examination, should have
+used MC003 for pMC004_b1</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/7/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1% Agarose gel run of PCR products from
+/6. Could not see products under blue light. Under UV light, products seemed
+more specific. Still not as strong as in previous gels. Perhaps need to
+troubleshoot PCR better. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_8"
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+v:shapes="Picture_x0020_8"><![endif]></span><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation Results</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Used iPhone application “<span
+class=SpellE>ColonyCount</span>” to assist in counting plates.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Plate Numbers are indicated on lower
+left of the pictures.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Average is 321 colonies.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
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+ style='width:540pt;height:960pt;visibility:visible;mso-wrap-style:square'>
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+</v:shape><![endif]--><![if !vml]><img border=0 width=720 height=1280
+src="..//wiki/images/8/8c/UChicago2015Notebook_filesimage018.jpg"
+alt="https://lh3.googleusercontent.com/X-VD4nUUhpipdr0rfrWJOvsNhjSK970Lyeqg4-7_LgaPEpds_4wfL591u_Zrf_LUL8d8TvSHd1p6C84HMcW1AoGnZqSuSnFUe2crNaJNiycNdr9dwgobKH7qmEoP5OK-XhGzmyc"
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+ style='width:540pt;height:960pt;visibility:visible;mso-wrap-style:square'>
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+v:shapes="Picture_x0020_12"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation Efficiency </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Used 1ul of 50pg/<span class=SpellE><span
+class=GramE>ul</span></span><span class=GramE> &nbsp;of</span> DNA </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>4: 287 / 5*10^-5 = 5.74*10^6 <span
+class=SpellE>cfu</span>/<span class=SpellE>ug</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>5: 328 / 5*10^-5 = 6.56*10^6 <span
+class=SpellE>cfu</span>/<span class=SpellE>ug</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>6: 414 / 5*10^-5 = 8.28*10^6 <span
+class=SpellE>cfu</span>/<span class=SpellE>ug</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>7: 254 / 5*10^-5 = 5.08*10^6 <span
+class=SpellE>cfu</span>/<span class=SpellE>ug</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Designed Sequencing primers as well as
+new primers for pMC001. Primers made specifically for oscillator plasmid
+-overhangs incorporated to make primers longer and more specific. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Prepared PCR of products seen on gel in
+morning (G1, G2, 001b1, 001b2, 004b1, used 1 <span class=SpellE>uL</span> of
+leftover PCR reaction). Used Q5 High Fidelity polymerase, as no <span
+class=SpellE>Phusion</span> available. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ingredients:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Q5
+High-Fidelity 2X Master Mix- 25 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DNA
+-1 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>F
+Primer -2.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>R
+Primer -2.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>H2O
+-19 <span class=SpellE>uL</span> </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings: 98C 30s, 98C
+s, 65C for 30s, 72C for 30s, 72C for 2mins, hold at 4C</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>30 cycles </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Prepared Colony PCR To confirm inserts
+of pMC004<span class=GramE>,5,6,7</span>. Used <span class=SpellE>Taq</span>
+DNA polymerase instead of <span class=SpellE>phusion</span>. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l19 level1 lfo27;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Pick single colony from plate, place in 50uL of dH2O (acts as DNA
+template)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l19 level1 lfo27;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add following PCR 1X Master Mix for <span class=SpellE>Taq</span>:<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>5 <span
+class=SpellE>uL</span> 10X buffer</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1 <span
+class=SpellE>uL</span> dNTPs</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1 <span
+class=SpellE>uL</span> 10 <span class=SpellE>uM</span> primer stock-VF2 and VR</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1 <span
+class=SpellE>uL</span> DNA stock</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>0.5 <span
+class=SpellE>uL</span> <span class=SpellE>Taq</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>41.5 <span
+class=SpellE>uL</span> H2O </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings: 95C 2mins, 95C
+s, 55C 15s,<span style='background:yellow'> 68C 45s</span> (30 cycles), 68C
+m, hold 4C</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>-&gt; Extend to 1min per kb (look up on
+product sheet)</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/9/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ran 1% Agarose gel 120V, 30 mins. Ran
+both Colony PCR and Q5 PCR. 5uL each sample loaded. Different ladder used
+(Quick Load Purple 2-Log from NEB. Same amount of EtBr (0.75 <span
+class=SpellE>uL</span>) used. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_13"
+ o:spid="_x0000_i1053" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/8ID1Wu4cCZQwLMrR4l7op7n00o_k_O7phTHC0tsU2gVfJ1M7bWLOpyTMvvGKtN7T6O0P9AAFRd9gPlqgh7bc7Ju2eNSnCx-p5oshQuhK0kr662ldgZoEARPb_2PHBhPCt0Xy-oc"
+ style='width:946.5pt;height:676.5pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/41/UChicago2015Notebook_filesimage025.png" o:title="8ID1Wu4cCZQwLMrR4l7op7n00o_k_O7phTHC0tsU2gVfJ1M7bWLOpyTMvvGKtN7T6O0P9AAFRd9gPlqgh7bc7Ju2eNSnCx-p5oshQuhK0kr662ldgZoEARPb_2PHBhPCt0Xy-oc"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1262 height=902
+src="..//wiki/images/a/a9/UChicago2015Notebook_filesimage026.png"
+alt="https://lh6.googleusercontent.com/8ID1Wu4cCZQwLMrR4l7op7n00o_k_O7phTHC0tsU2gVfJ1M7bWLOpyTMvvGKtN7T6O0P9AAFRd9gPlqgh7bc7Ju2eNSnCx-p5oshQuhK0kr662ldgZoEARPb_2PHBhPCt0Xy-oc"
+v:shapes="Picture_x0020_13"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/10/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel repeated, this time using leftover
+uL of sample. 1kB Plus <span class=SpellE>invitrogen</span> ladder used. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_14"
+ o:spid="_x0000_i1052" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/DIjzha_kO7vq-_NlZnE4aQiO-9rI8Ja38iqhsjQvnnfAiNpJ9t1qBoxcP-r28_jiOA9_tk_W3HHrGTDFducvEWnD0wUejHXyfH7VIUQ8_gsQzq850CGEVFKEiOwND6aZm-xL6Q4"
+ style='width:820.5pt;height:750.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/a/ab/UChicago2015Notebook_filesimage027.png" o:title="DIjzha_kO7vq-_NlZnE4aQiO-9rI8Ja38iqhsjQvnnfAiNpJ9t1qBoxcP-r28_jiOA9_tk_W3HHrGTDFducvEWnD0wUejHXyfH7VIUQ8_gsQzq850CGEVFKEiOwND6aZm-xL6Q4"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1094 height=1001
+src="..//wiki/images/9/9d/UChicago2015Notebook_filesimage028.png"
+alt="https://lh4.googleusercontent.com/DIjzha_kO7vq-_NlZnE4aQiO-9rI8Ja38iqhsjQvnnfAiNpJ9t1qBoxcP-r28_jiOA9_tk_W3HHrGTDFducvEWnD0wUejHXyfH7VIUQ8_gsQzq850CGEVFKEiOwND6aZm-xL6Q4"
+v:shapes="Picture_x0020_14"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Innoculation</span></b></span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>-</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Due to failure of Colony PCR, colonies
+were inoculated and incubated. Will conduct direct <span class=SpellE>miniprep</span>
+on 8/11 and sequence to sequence. This should help in determining if there is a
+problem with primers/insert or with the PCR. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson assembly of BH001_b1 and <span
+class=SpellE>Cam</span> backbone conducted. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_15"
+ o:spid="_x0000_i1051" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/Ro40wlzRHwUwwISfK3r4r6RoQEsg9yIQboeqcm1iqJCQx6qSgQSqdzstLBpIWRx43kS_3ETyfw-RNlrAxvFvBgeVPu5Vs-X8EOlc_n6NdLlav5_rhGWSgjOW3V3m5hTqwUWKvaA"
+ style='width:997.5pt;height:66.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/46/UChicago2015Notebook_filesimage029.png" o:title="Ro40wlzRHwUwwISfK3r4r6RoQEsg9yIQboeqcm1iqJCQx6qSgQSqdzstLBpIWRx43kS_3ETyfw-RNlrAxvFvBgeVPu5Vs-X8EOlc_n6NdLlav5_rhGWSgjOW3V3m5hTqwUWKvaA"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1330 height=89
+src="..//wiki/images/1/15/UChicago2015Notebook_filesimage030.png"
+alt="https://lh3.googleusercontent.com/Ro40wlzRHwUwwISfK3r4r6RoQEsg9yIQboeqcm1iqJCQx6qSgQSqdzstLBpIWRx43kS_3ETyfw-RNlrAxvFvBgeVPu5Vs-X8EOlc_n6NdLlav5_rhGWSgjOW3V3m5hTqwUWKvaA"
+v:shapes="Picture_x0020_15"><![endif]></span><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR-</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up PCR for <span class=SpellE>biobricks</span>
+MC002, and MC003 and BH001 (confirmation). &nbsp;</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>11X PCR Master Mix</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Master Mix Recipe-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 110 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 11 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 5.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 341 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 16.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Set up PCR for biobricks MC002, and MC003 and BH001 (confirmation).  </p>
+mso-bidi-font-family:Arial;color:black'>44uL of Master <span class=GramE>mix</span>,
-<p>11X PCR Master Mix</p>
+.5 <span class=SpellE>uL</span> of F primer, 2.5 <span class=SpellE>uL</span>
-<p>PCR Master Mix Recipe-</p>
+of R Primer and 1 <span class=SpellE>uL</span> of template DNA used. </span><span
-<p>o   Phusion 5X HF Buffer =3D 110 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>o   dNTPs 10 mM  =3D 11 uL</p>
-<p>o   Phusion DNAP =3D 5.5 uL</p>
-<p>o   H2O =3D 341 uL</p>
-<p>o   DMSO =3D 16.5 uL</p>
-<p>44uL of Master mix, 2.5 uL of F primer, 2.5 uL of R Primer and 1 uL of template DNA used. </p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<table>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
-<tr>
+ style='width:6.5in;border-collapse:collapse;mso-yfti-tbllook:1184'>
-   <th>DNA Template</th>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
-   <th>Primers</th>
+   <td valign=top style='border:solid black 1.0pt;mso-border-alt:solid black .75pt;
-   <th>Info</th>
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
-   <th>Gel to Run</th>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
-</tr><tr>
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<td>MC002_b1</td><td><p>MC029, MC010</p><p>1815bps</p></td><td>Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts</td><td>1% Agarose</td>
+  mso-bidi-font-family:Arial;color:black'>DNA Template</span></b><span
-</tr><tr>
+   style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
-<td>MC003_b1</td><td><p>MC029, MC014</p><p>1815 bps</p></td><td>Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts</td><td>1% Agarose</td>
+  "Times New Roman"'><o:p></o:p></span></p>
-</tr><tr>
+  </td>
-<td>MC008_b1</td><td><p>MC003, MC028</p><p>1294 bps</p></td><td>Clone out kaibc promoter/GFP</td><td>1% Agarose</td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
-</tr><tr>
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
-<td>Biobrick B0015 Terminator</td><td><p>MC027, MC030</p><p>129 bps</p></td><td>Clone out terminator</td><td>2% Agarose</td>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
-</tr><tr>
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<td>BH001/Cam  Gibson</td><td><p>MC003, MC004</p><p>765 bps</p><p>*should not have done</p></td><td>? Was to clone out insert, should have transformed as is. </td><td>1% Agarose</td>
+  mso-bidi-font-family:Arial;color:black'>Primers</span></b><span
-</tr>
+   style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>Info</span></b><span
+   style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>Gel to Run</span></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>MC002_b1</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC029, MC010</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1815
+  bps</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Clone
+  out b1 to give right initial sequence for <span class=SpellE>kaibc</span>/GFP/Term
+  inserts</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1%
+  Agarose</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>MC003_b1</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC029, MC014</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1815
+  bps</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Clone
+  out b1 to give right initial sequence for <span class=SpellE>kaibc</span>/GFP/Term
+  inserts</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1%
+  Agarose</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>MC008_b1</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC003, MC028</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1294
+  bps</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Clone
+  out <span class=SpellE>kaibc</span> promoter/GFP</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1%
+  Agarose</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span class=SpellE><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:black'>Biobrick</span></span><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'> B0015
+  Terminator</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC027, MC030</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>129
+  bps</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Clone
+  out terminator</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2%
+  Agarose</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>BH001/Cam
+  &nbsp;Gibson</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC003, MC004</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>765 bps</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>*should
+  not have done</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>?
+  Was to clone out insert, should have transformed as is. </span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1%
+  Agarose</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
 </table>
-<p>Thermocycler settings- 98C 2min, 98C 15s, 67C 30s, 72C 1.5 mins, 72C 10min, 4 hold. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98C 2min, 98C
+s, 67C 30s, 72C 1.5 mins, 72C 10min, 4 hold. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/11/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>2% gel for Terminator cloning sample
+and 1% gel for other PCR samples (see table) run. 120V for 30 min. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_16" o:spid="_x0000_i1050"
+ type="#_x0000_t75" alt="https://lh4.googleusercontent.com/LLWlJ3IaCAGROIYBCESSzLg07w49CbpqwO-jiHPXXg_0jTytoaCrpadQQqW9zWtMe6M_d8ai66I1cO0xIZB_q0sjpsYZe5GlQXv6wmApYvj0oYX-c7SnkaaDVsajcRbmUOkwWrM"
+ style='width:431.25pt;height:228pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/47/UChicago2015Notebook_filesimage031.png" o:title="LLWlJ3IaCAGROIYBCESSzLg07w49CbpqwO-jiHPXXg_0jTytoaCrpadQQqW9zWtMe6M_d8ai66I1cO0xIZB_q0sjpsYZe5GlQXv6wmApYvj0oYX-c7SnkaaDVsajcRbmUOkwWrM"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=575 height=304
+src="..//wiki/images/f/fb/UChicago2015Notebook_filesimage032.png"
+alt="https://lh4.googleusercontent.com/LLWlJ3IaCAGROIYBCESSzLg07w49CbpqwO-jiHPXXg_0jTytoaCrpadQQqW9zWtMe6M_d8ai66I1cO0xIZB_q0sjpsYZe5GlQXv6wmApYvj0oYX-c7SnkaaDVsajcRbmUOkwWrM"
+v:shapes="Picture_x0020_16"><![endif]><!--[if gte vml 1]><v:shape id="Picture_x0020_17"
+ o:spid="_x0000_i1049" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/usfO-NmUO1TQvxoXM3nWF115qobDFPP21ORF7afn0YMDNK45AGLAfejyjeuwAIkz6WbeigWbHlrF40zR0YKnEye-Br6cvgQY4S3rzPZNPPr_ysLLbqL-05XSAI-J3KEYsljmmAs"
+ style='width:180pt;height:269.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage033.png" o:title="usfO-NmUO1TQvxoXM3nWF115qobDFPP21ORF7afn0YMDNK45AGLAfejyjeuwAIkz6WbeigWbHlrF40zR0YKnEye-Br6cvgQY4S3rzPZNPPr_ysLLbqL-05XSAI-J3KEYsljmmAs"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=240 height=359
+src="..//wiki/images/4/46/UChicago2015Notebook_filesimage034.png"
+alt="https://lh4.googleusercontent.com/usfO-NmUO1TQvxoXM3nWF115qobDFPP21ORF7afn0YMDNK45AGLAfejyjeuwAIkz6WbeigWbHlrF40zR0YKnEye-Br6cvgQY4S3rzPZNPPr_ysLLbqL-05XSAI-J3KEYsljmmAs"
+v:shapes="Picture_x0020_17"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Miniprep</span></b></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/11/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH protocol</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>2% gel for Terminator cloning sample and 1% gel for other PCR samples (see table) run. 120V for 30 min. </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel12.png]]
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
-[[File:UChicagoGel13.png]]
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<html>
-<p><b>Miniprep</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>BH protocol</b></p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><u><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>PCR of <span class=SpellE>Minipreps</span></span></u><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b><u>PCR of Minipreps</u></p>
+mso-bidi-font-family:Arial;color:black'>(BH protocol)</span></u><span
-<p>(BH protocol)</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	PCR of MC002/3 blocks</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Phusion 7 x of 1X mix</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>	HF Buffer -70 uL</p>
-<p>	DMSO -10.5 uL</p>
-<p>	DNTPs -7 uL </p>
-<p>	H2O -219 uL</p>
-<p>	Phusion DNAP -3.5 uL</p>
-<p>Use 44 uL of master mix w/ 2.5 of F and R primers and 1 uL of template DNA.</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span><u>PCR
+of MC002/3 blocks</u></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>DNA Template</b></p><p><b>Primers</b></p><p>MC002_b1</p><p>MC029, MC010</p><p>1815 bps product</p><p>MC003_b1</p><p>MC029, MC014</p><p>1815 bps product</p><p>MC008_b1</p><p>MC003, MC028</p><p>1294 bps product</p>
+<p class=MsoNormal style='margin-left:1.0in'><span class=SpellE><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Phusion</span></span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'> 7 x of 1X mix</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Thermocycler Settings-</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds</p><p>30 Cycles</p><p>98=C2=B0C</p><p>65=C2=B0C</p><p>72=C2=B0C</p><p>10 seconds</p><p>30 seconds</p><p>1 min (30s x 1.8kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>HF Buffer -70 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Thermocycler Settings for Mini-Prep PCR-</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>120 seconds</p><p>30 Cycles</p><p>98=C2=B0C</p><p>61.6=C2=B0C </p><p>(NEB - DMSO%*0.8)</p><p>72=C2=B0C</p><p>15 seconds</p><p>30 seconds</p><p>1 min (30s x 1.8kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>5 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DMSO -10.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DNTPs -7 <span class=SpellE>uL</span> </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>H2O -219 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span><span class=SpellE>Phusion</span> DNAP
+-3.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/12/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>PCR of Terminator </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use 44 <span class=SpellE>uL</span> of
+master mix w/ 2.5 of F and R primers and 1 <span class=SpellE>uL</span> of
+template DNA.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>2 50 uL Samples  </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>	HF Buffer -10 uL</p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
-<p>	DMSO -1.5 uL</p>
+ style='width:6.5in;border-collapse:collapse;mso-yfti-tbllook:1184'>
-<p>	DNTPs -1 uL </p>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
-<p>	H2O -31 uL</p>
+  <td valign=top style='border:solid black 1.0pt;mso-border-alt:solid black .75pt;
-<p>	Phusion DNAP -0.5 uL</p>
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
-<p>	10 uM of MC027 Primer -2.5 uL </p>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
-<p>	10 uM MC030 Primer -2.5 uL</p>
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	DNA from plate -1 uL</p>
+  mso-bidi-font-family:Arial;color:black'>DNA Template</span></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>Primers</span></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:black'>MC002_b1</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC029, MC010</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1815
+  bps product</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:black'>MC003_b1</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC029, MC014</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1815
+  bps product</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center;mso-line-height-alt:
+pt'><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:black'>MC008_b1</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:black'>MC003, MC028</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1294
+  bps product</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-<p>Thermocycler Settings: 1 cycle: 98C for 2 mins, 5 cycles: 98C for 15s, 69C for 30s, 72C for 2 min 30 cycles: 98C for 15s, 72C for 1.5 min, 1 cycle 72C for 10 min, 4C hold. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>50 mL 2% gel, 150 mL 1% gel, and 100 mL 1% gel run for Miniprep PCR as well as MC002/3 clone parts PCR. 25 uL of sample loaded for Terminator 2%  gel. 19 uL sample loaded for 150mL Miniprep gel. 45 uL of sample loaded for MC002/3 clone parts 1% gel.</p>
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-[[File:UChicagoGel14.png]]
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
-<html>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>65°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1 min (30s x 1.8kb -largest product)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-<p>1kB plus Invitrogen ladder used. Send samples 41,43,51,52,53,54,61,62,63,64,74 for sequencing. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Images:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings for Mini-Prep
+PCR-</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-[[File:UChicagoGel15.png]]
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
-[[File:UChicagoGel16.png]]
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
-<html>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>120 seconds</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>61.6°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>(NEB - DMSO%*0.8)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>15 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1 min (30s x 1.8kb -largest product)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>5 min</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-<p><b>Gel Purification-</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>Extracted correctly sized product fragmnets under blue light: T-189 bps, GFP 1249 bps. </p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<p>Weights of gels:</p>
+mso-bidi-font-family:"Times New Roman"'><br>
-<p>GFP 1-63.8 mg</p>
+<br style='mso-special-character:line-break'>
-<p>GFP 2-70.64 mg</p>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
-<p>T 1-34.6 mg</p>
+<![endif]><o:p></o:p></span></p>
-<p>T 2-110.0 mg</p>
-<p>Used Machery-Nagel clean up method:</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<ol><li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+mso-bidi-font-family:Arial;color:black'>8/12/15</span></b><span
-<li>Incubate at 50C for 10 mins</li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Transfer solution to spin column and collection tube. </li>
-<li>Spin at 11,000g (RCFs) for 30s</li>
-<li>Discard flow through. Add 700 uL of wash buffer NT3 to spin column.</li>
-<li>Spin at 11,000g (RCFs) for 30s</li>
-<li>Discard flow through. Add 700 uL of wash buffer NT3 to spin column. </li>
-<li>Spin at 11,000g (RCFs) for 30s</li>
-<li>Spin at 11,000g (RCFs) for 1 min to dry silica membrane </li>
-<li>Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 30s</li>
-<li>Nanodrop (use EB buffer as blank, load 1.5 uL sample)</li></ol>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Purity recorded w/Nanodrop:</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:.5in'><u><span
-<p>T1- 5.1ng/uL 260/280-14.84</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<p>T2-23.1ng/uL 260/280-1.92</p>
+color:black'>PCR of Terminator </span></u><span style='font-size:16.0pt;
-<p>GFP 1- 41.6ng/uL 260/280-1.94</p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>GFP 2- 33.1ng/uL 260/280-2.03</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Plan for MC002_b1, MC003_b1: </b>There is low yield of insert probably due to repeating regions of DNA in blocks 1 of MC002 and MC003. As IDT expressed, there are a lot of low mass products that are more efficiency amplified by primers. Therefore, as Jennifer Moran mentioned, the best course of action would be to insert these two gblocks into a Zero Blunt TOPO PCR Vector and transforming into competent cells to amplify our gblocks. After producing colonies, we can PCR and then screen for colonies with correct product size. We will then miniprep and send these blocks in for sequencing. This will take more time, but will ensure the purity of the insert. After confirming the correct sequence, the DNA from the miniprep/gel extraction? can be used to gibson the GFP/kaibc, the terminator, and the first blocks together. </p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:.5in'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>2 50 <span class=SpellE>uL</span> Samples &nbsp;</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>HF Buffer -10 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DMSO -1.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Mini-Prep Results (Nano-Drop)</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DNTPs -1 <span class=SpellE>uL</span> </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>H2O -31 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Sample</p><p>260/280</p><p>260/230</p><p>ng/ul</p><p>41</p><p>1.86</p><p>2.20</p><p>55.1</p><p>42</p><p>1.84</p><p>2.06</p><p>35.4</p><p>43</p><p>1.89</p><p>2.10</p><p>72.9</p><p>44</p><p>1.92</p><p>1.83</p><p>30.7</p><p>51</p><p>1.94</p><p>1.87</p><p>46.8</p><p>52</p><p>1.90</p><p>2.00</p><p>53.9</p><p>53</p><p>1.90</p><p>2.15</p><p>66.6</p><p>54</p><p>1.93</p><p>2.14</p><p>58.6</p><p>61</p><p>1.97</p><p>2.06</p><p>52.2</p><p>62</p><p>1.97</p><p>2.14</p><p>61.1</p><p>63</p><p>2.00</p><p>2.24</p><p>52.5</p><p>64</p><p>1.92</p><p>2.12</p><p>57.1</p><p>71</p><p>2.03</p><p>2.13</p><p>56.9</p><p>72</p><p>1.88</p><p>1.81</p><p>50.2</p><p>73</p><p>1.93</p><p>1.83</p><p>59.3</p><p>74</p><p>1.92</p><p>1.92</p><p>53.3</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span><span class=SpellE>Phusion</span> DNAP
+-0.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>The highlighted ones are the ones that we choose to sequence.</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>10 <span class=SpellE>uM</span> of
+MC027 Primer -2.5 <span class=SpellE>uL</span> </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>10 <span class=SpellE>uM</span> MC030
+Primer -2.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DNA from plate -1 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Sequencing-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>The concentration of the DNA templates were too low, so we used 10 ug of each.</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>The primers were diluted to a 4uM solution from a 100x stock.</p>
-<p>(1.4 ul of primer + 33.6 ul of water) </p>
-<p>VF2 [1]</p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
-<p>MC003 (F) [2]</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Thermocycler
-<p>MC041 (F) [3]</p>
+Settings: 1 cycle: 98C for 2 mins, 5 cycles: 98C for 15s, 69C for 30s, 72C for
-<p>MC023 (F) [4]</p>
+min 30 cycles: 98C for 15s, 72C for 1.5 min, 1 cycle 72C for 10 min, 4C hold.
-<p>MC022 (R) [5]</p>
+</span><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
-<p>VR [6]</p>
+"Times New Roman"'><o:p></o:p></span></p>
-<p>[41]</p>
-<p>[43]</p>
-<p>[53]</p>
-<p>[54]</p>
-<p>[62]</p>
-<p>[63]</p>
-<p>[71]</p>
-<p>[73]</p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoTable3.png]]
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<html>
-<p>^Things sent.</p>
+<p class=MsoNormal style='margin-left:.5in'><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Gel
+Electrophoresis-</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Transformation of BH001:</b></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
-<p>DNA was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>50 mL 2%
+gel, 150 mL 1% gel, and 100 mL 1% gel run for <span class=SpellE>Miniprep</span>
+PCR as well as MC002/3 clone parts PCR. 25 <span class=SpellE>uL</span> of
+sample loaded for Terminator 2<span class=GramE>% &nbsp;gel</span>. 19 <span
+class=SpellE>uL</span> sample loaded for 150mL <span class=SpellE>Miniprep</span>
+gel. 45 <span class=SpellE>uL</span> of sample loaded for MC002/3 clone parts
+% gel. 1kB plus Invitrogen ladder used. Send samples 41,43,51,52,53,54,61,62,63,64,74
+for sequencing. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
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+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Remove cells from freezer, incubate tubes on ice </li>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Add 1 uL DNA (50 pg//uL) into competent cell tube </li>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<li>Incubate tube on ice for 30 mins</li>
-<li>Incubate tube 42C water bath for 1 min heat shock</li>
-<li>Incubate tube ice 5 mins </li>
-<li>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</li>
-<li>Incubate tube in shaker at 37C/1100 RPM for 1 hour</li>
-<li>Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking</li>
-<li>Collect pellet, spin 3000 rcf/gs for 3 mins </li>
-<li>Decant 800 uL of supernatant </li>
-<li>Use glass beads (5-6) per section (use sterile flame)</li>
-<li>Mix pellet, pipetted 200 uL in total</li>
-<li>Shake with beads and remove </li>
-<li>Incubate plate overnight 37C</li></ol>
+<p class=MsoNormal style='margin-left:.5in'><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Images:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/13/15-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
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+v:shapes="Picture_x0020_20"><![endif]></span><o:p></o:p></span></p>
-</html>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-[[File:UChicagoPlate2.png]]
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
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+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;mso-border-alt:solid black .25pt;
+  padding:.75pt .75pt .75pt .75pt'></td>
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+  solid black .25pt;mso-border-alt:solid black .25pt;padding:.75pt .75pt .75pt .75pt'></td>
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+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:.75pt .75pt .75pt .75pt'></td>
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+  solid black .25pt;mso-border-alt:solid black .25pt;padding:.75pt .75pt .75pt .75pt'></td>
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+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:.75pt .75pt .75pt .75pt'></td>
+ </tr>
+ <tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:.75pt .75pt .75pt .75pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:.75pt .75pt .75pt .75pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:.75pt .75pt .75pt .75pt'></td>
+ </tr>
+</table>
-<p>Transformation Efficiency of BH001-</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>amt dna used/plate</p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
-<p>Plates 1 and 2 were discarded, a colony PCR was done on 6 samples from Plate 3</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Purification-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>They were PCRed separately with two different annealing temperatures, 61.6 and 66. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Extracted correctly sized product <span
+class=SpellE>fragmnets</span> under blue light: T-189 bps, GFP 1249 bps. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gibson Assembly</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gibson assembly using 5uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hour 50C heatblock.</p>
+mso-bidi-font-family:Arial;color:black'>Weights of gels:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>GFP 1-63.8 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Prepared 2 50uL 1X PCR samples. </p>
+mso-bidi-font-family:Arial;color:black'>GFP 2-70.64 mg</span><span
-<p>	HF Buffer -10 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	DMSO -1.5 uL</p>
-<p>	DNTPs -1 uL </p>
-<p>	H2O -31 uL</p>
-<p>	Phusion DNAP -0.5 uL</p>
-<p>	F Primer 10 uM MC003 -2.5 uL</p>
-<p>	R Primer 10uM MC004 -2.5 uL </p>
-<p>	DNA (Gibson assembly rxn 8/13) -1uL </p>
-<p>Thermocycler Settings-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T 1-34.6 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds </p><p>30 Cycles</p><p>98=C2=B0C</p><p>65=C2=B0C</p><p>72=C2=B0C</p><p>10 seconds</p><p>30 secondsc</p><p>1.65 mins (30s x 3.3kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T 2-110.0 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Thermocycler Settings for Mini-Prep PCR- Low Temp</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>120 seconds</p><p>30 Cycles</p><p>98=C2=B0C</p><p>61.6=C2=B0C </p><p>(NEB - DMSO%*0.8)</p><p>72=C2=B0C</p><p>15 seconds</p><p>30 seconds</p><p>1 min (30s x 1.8kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>5 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Thermocycler Settings for Mini-Prep PCR- High Temp</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>120 seconds</p><p>30 Cycles</p><p>98=C2=B0C</p><p>66=C2=B0C </p><p>72=C2=B0C</p><p>15 seconds</p><p>30 seconds</p><p>1 min (30s x 1.8kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>5 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-bidi-font-family:Arial;color:black'>Used <span class=SpellE>Machery</span>-Nagel
+clean up method:</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<p><b>	</b>100 mL of 1% agarose gel run 120V, 30 mins. Samples loaded inclde 45 uL of BH003 biobrick and 20 uL of colony PCRs. 5 uL 1 kB plus Invitrogen ladder loaded. Colony PCRs yielded no results, biobrick BH003 extracted using gel punches (seen in image). First column of BH3 seemed to give very low yield (light band not strong band seen before extraction).</p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 200uL NTI Binding buffer / 100 mg gel <o:p></o:p></span></p>
-<p><b>Image-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate at 50C for 10 mins<o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-[[File:UChicagoGel17.png]]
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Transfer solution to spin column and collection tube. <o:p></o:p></span></p>
-<p><b>Gel Extraction-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<p>BH003 biobrick extracted from gel. Machery-Nagel protocol used for gel extraction. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<p>Weight of gel: 85.2 mg</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p>Used Machery-Nagel clean up method:</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<li>Incubate at 50C for 10 mins</li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
-<li>Transfer solution to spin column and collection tube. </li>
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
-<li>Spin at 11,000g (RCFs) for 30s</li>
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-<li>Discard flow through. Add 700 uL of wash buffer NT3 to spin column.</li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<li>Spin at 11,000g (RCFs) for 30s</li>
+color:black'>Discard flow through. Add 700 <span class=SpellE>uL</span> of wash
-<li>Discard flow through. Add 700 uL of wash buffer NT3 to spin column. </li>
+buffer NT3 to spin column.<o:p></o:p></span></p>
-<li>Spin at 11,000g (RCFs) for 30s</li>
-<li>Spin at 11,000g (RCFs) for 1 min to dry silica membrane </li>
-<li>Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 30s</li>
-<li>Nanodrop (use EB buffer as blank, load 1.5 uL sample)</li></ol>
-<p>Nanodrop purity- 14.4 ng/uL, 260/280- 1.55</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>6.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<p><b>	</b>Made 4 1X 50uL samples to PCR ampicillin backbone. Used 1 uL of 25ng/uL amp linearized backbone. Primers MC001 and MC002. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>7.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Discard flow through. Add 700 <span class=SpellE>uL</span> of wash
+buffer NT3 to spin column. <o:p></o:p></span></p>
-<p>PCR Master Mix Recipe-</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<p>o   Phusion 5X HF Buffer =3D 50 uL</p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<p>o   dNTPs 10 mM  =3D 5  uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
-<p>o   Phusion DNAP =3D 2.5 uL</p>
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>8.<span
-<p>o   H2O =3D 155 uL</p>
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
-<p>o   DMSO =3D 7.5 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
-<p>o   DNA (products from Gibson 8/4)=3D 1.0uL</p>
+color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p>o   F Primer MC001=3D 2.5uL</p>
-<p>o   R Primer MC002 =3D2.55uL</p>
-<p>Thermocycler settings:98C 30s, 25 cycles: 98C 10s, 69C 30s, 72C 1.5m, 72.0C 10 min, 4C hold</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>9.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Spin at 11,000g (RCFs) for 1 min to dry silica membrane <o:p></o:p></span></p>
-<p><b>Gibson Assembly-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-<p><b>	</b>Given success of gibson and transformation with BH001, decided to try assemble pMC001 and transform directly. Reaction incubated on heatblock for 1 hour at 50C. </p>
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>10.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Elute DNA with 15 <span class=SpellE>uL</span>
+of NE elution buffer. Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l21 level1 lfo28;
-[[File:UChicagoTable4.png]]
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>11.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span class=SpellE><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> (use EB buffer as blank, load 1.5 <span class=SpellE>uL</span>
+sample)<o:p></o:p></span></p>
-<p>	</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>50 mL of 1% agarose gel made to run amp backbone amplifications. 0.5 uL EtBr used. Gel run 120V for 30 mins. 1 kB Plus Invitrogen ladder used. 45 uL of samples loaded. </p>
+mso-bidi-font-family:Arial;color:black'>Purity recorded w/<span class=SpellE>Nanodrop</span>:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T1- 5.1ng/<span class=SpellE>uL</span>
+/280-14.84</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image- </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T2-23.1ng/<span class=SpellE>uL</span>
+/280-1.92</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel18.png]]
+mso-bidi-font-family:Arial;color:black'>GFP 1- 41.6ng/<span class=SpellE>uL</span>
-<html>
+/280-1.94</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Extraction and Purification-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Ampicillin backbone  extracted from gel. Machery-Nagel protocol used for gel extraction. </p>
+mso-bidi-font-family:Arial;color:black'>GFP 2- 33.1ng/<span class=SpellE>uL</span>
-<p>Weight of gel samples:</p>
+/280-2.03</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A1 -135.8 mg (271.6 uL of binding buffer NTI used)</p>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>A2 -201.4mg (402.8 uL of binding buffer NTI used) </p>
-<p>Nanodrop concentrations of ampicillin backbone samples</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>A1 - 32.3 ng/uL, 260/280- 1.84</p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<p>A2 - 33.4 ng/uL, 260/280- 1.59 </p>
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Gibson-</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gibson assembly of ampicillin backbone to BH003 insert conducted. Used 5uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hour 50C heatblock.</p>
+mso-bidi-font-family:Arial;color:black'>Plan for MC002_b1, MC003_b1: </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>There is low yield of insert probably due to repeating regions of
+DNA in blocks 1 of MC002 and MC003. As IDT expressed, there are a lot of low
+mass products that are more efficiency amplified by primers. Therefore, as
+Jennifer Moran mentioned, the best course of action would be to insert these
+two <span class=SpellE>gblocks</span> into a Zero Blunt TOPO PCR Vector and
+transforming into competent cells to amplify our <span class=SpellE>gblocks</span>.
+After producing colonies, we can PCR and then screen for colonies with correct
+product size. We will then <span class=SpellE>miniprep</span> and send these
+blocks in for sequencing. This will take more time, but will ensure the purity
+of the insert. After confirming the correct sequence, the DNA from the <span
+class=SpellE>miniprep</span>/gel extraction? <span class=GramE>can</span> be
+used to <span class=SpellE>gibson</span> the GFP/<span class=SpellE>kaibc</span>,
+the terminator, and the first blocks together. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-[[File:UChicagoTable7.png]]
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<html>
+mso-bidi-font-family:"Times New Roman"'><br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
-<p><b>Transformation of BH003:</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>DNA was transformed into competent cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></p>
+mso-bidi-font-family:Arial;color:black'>Mini-Prep Results (Nano-Drop)</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Remove cells from freezer, incubate tubes on ice </li>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<li>Add 1 uL DNA (50 pg//uL) into competent cell tube </li>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<li>Incubate tube on ice for 30 mins</li>
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<li>Incubate tube 42C water bath for 1 min heat shock</li>
-<li>Incubate tube ice 5 mins </li>
-<li>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</li>
-<li>Incubate tube in shaker at 37C/1100 RPM for 1 hour</li>
-<li>Heat Cam plate in incubator as cold plate reduces efficiency,complete while cells shaking</li>
-<li>Collect pellet, spin 3000 rcf/gs for 3 mins </li>
-<li>Decant 800 uL of supernatant </li>
-<li>Use glass beads (5-6) per section (use sterile flame)</li>
-<li>Mix pellet, pipetted 200 uL in total</li>
-<li>Shake with beads and remove </li>
-<li>Incubate plate overnight 37C</li></ol>
-<p><b>8/14/15</b></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
+ style='width:6.5in;border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Sample</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>260/280</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>260/230</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>ng/<span
+  class=SpellE>ul</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>41</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.86</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.20</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>55.1</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>42</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.84</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2.06</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>35.4</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>43</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.89</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.10</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>72.9</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>44</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.92</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.83</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>30.7</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:5'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>51</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.94</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.87</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>46.8</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:6'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>52</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.90</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2.00</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>53.9</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:7'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>53</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.90</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.15</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>66.6</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:8'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>54</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.93</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.14</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>58.6</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:9'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>61</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.97</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.06</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>52.2</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:10'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>62</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.97</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.14</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>61.1</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:11'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>63</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2.00</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2.24</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>52.5</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:12'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>64</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.92</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>2.12</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>57.1</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:13'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>71</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.03</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>2.13</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>56.9</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:14'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>72</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.88</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.81</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>50.2</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:15'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>73</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.93</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>1.83</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black;
+  background:#FFE599'>59.3</span><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:16;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .75pt;mso-border-alt:solid black .75pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>74</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.92</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1.92</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .75pt;
+  mso-border-left-alt:solid black .75pt;mso-border-alt:solid black .75pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>53.3</span><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>The highlighted ones are the ones that
+we choose to sequence.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Transformation-</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>	MC001 and BH003 gave good results from transformation. Colonies were small, but evenly distributed. Colonies could be small because plates incubated late last night. </p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoPlate5.png]]
+mso-bidi-font-family:Arial;color:black'>Sequencing-</span></b><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Colony PCR of BH003/MC001-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Diluted one colony from plates with most growth into 50uL of dH2O to serve as DNA template. </p>
+mso-bidi-font-family:Arial;color:black'>The concentration of the DNA templates
-<p>Made 8.5 x of 1X PCR Master Mix:</p>
+were too low, so we used 10 <span class=SpellE>ug</span> of each.</span><span
-<p>	HF Buffer: 85 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	DMSO: 12.75 uL</p>
-<p>	dNTPS: 8.5 uL</p>
-<p>	H2O: 263.5 uL</p>
-<p>	DNAP: 4.25 uL</p>
-<p>	</p>
-<p>	Used 2.5uL of VF2 and VR each and 1 uL of sample DNA. </p>
-<p>Thermocycler Settings for Colony </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>10 minutes </p><p>30 Cycles</p><p>98=C2=B0C</p><p>66=C2=B0C </p><p>72=C2=B0C</p><p>15 seconds</p><p>30 seconds</p><p>2.5 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-bidi-font-family:Arial;color:black'>The primers were diluted to a 4uM
+solution from a 100x stock.</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><blockquote>Sequencing Primers for MC001- MC003,MC031,MC032,MC006,MC033, MC008</blockquote></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>(1.4 <span class=SpellE>ul</span> of
+primer + 33.6 <span class=SpellE>ul</span> of water) </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/15/</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis </b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>	</b>1% Agarose gel run for colony PCR. 1 kB Invitrogen plus ladder used. 50 uL of sample loaded. </p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel19.png]]
+mso-bidi-font-family:Arial;color:black'>VF2 [1]</span><span style='font-size:
-<html>
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/17</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Sequences Submitted</b></p>
+mso-bidi-font-family:Arial;color:black'>MC003 (F) [2]</span><span
-<p>MC001 note submitted on weekend for sequencing. MC001 submitted in morning for sequencing. 4uM primer stock made by diluting in water (1:24 primer:water ratio). 14 uL of miniprepped DNA submitted with 10uL of 4uM primers. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Primers used: </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>VF2 (1)</p>
+mso-bidi-font-family:Arial;color:black'>MC041 (F) [3]</span><span
-<p>VR (2)</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>MC003 (3)</p>
-<p>MC031 (4)</p>
-<p>MC032 (5)</p>
-<p>MC006 (6)</p>
-<p>MC033 (7)</p>
-<p>MC008 (8) </p>
-<p><b>Colony PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Colony PCR of MC001 set up again as upon closer examination, insert seems too small to be correct. </p>
+mso-bidi-font-family:Arial;color:black'>MC023 (F) [4]</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Diluted one colony from plates with most growth into 50uL of dH2O to serve as DNA template. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2 uL DNA):</p>
+mso-bidi-font-family:Arial;color:black'>MC022 (R) [5]</span><span
-<p>	HF Buffer: 18.00</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	DMSO: 2.70 uL</p>
-<p>	dNTPS: 1.80</p>
-<p>	H2O: 55.</p>
-<p>	DNAP: 4.25 uL</p>
-<p>	Used 4.50 uL of 10X MC003 and MC004 for mix. </p>
-<p>Thermocycler Settings for Colony </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>10 minutes </p><p>30 Cycles</p><p>98=C2=B0C</p><p>66=C2=B0C </p><p>72=C2=B0C</p><p>15 seconds</p><p>30 seconds</p><p>2.5 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-bidi-font-family:Arial;color:black'>VR [6]</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><blockquote>Talked with Justin about ordering materials -TOPO kit should come tomorrow. </blockquote></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>[41]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/18</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis-</b></p>
+mso-bidi-font-family:Arial;color:black'>[43]</span><span style='font-size:16.0pt;
-<p>0.8% (to separate out larger fragments with more efficiency) agarose gel cast. Gel run on 120V for 30 mins. 1kB plus Invitrogen ladder used. No results from colony PCR, indicating something wrong during PCR. Likely issue with primers? =C2=BE Used instead of VF2 and VR. Have double checked primers are correct -could be issue with insert and primers. Note -issues with evaporation in thermocycler, which is why samples 115,116,117,118,128 could not be loaded. </p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>[53]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel20.png]]
+mso-bidi-font-family:Arial;color:black'>[54]</span><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Sequencing Results</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Sequencing successful for MC004,5,7. MC006 and MC007 samples probably mislabelled as sequencing indicates correct MC006 phosphomimetic is in MC007 sequence and vice versa. Otherwise, MC007,5, and 4 are all correctly sequenced. Point mutation in MC006 (labelled MC007) samples base 244 of Snapgene file. Point mutation seems to be silent (GGC to GGT, translate features lists both as glycine. Four read out KaiC variants seem good to go. </p>
+mso-bidi-font-family:Arial;color:black'>[62]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>[63]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>MC001 -still waiting for colony PCR/ specific primers to arrive for assembly</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>MC002/3 -still waiting for TOPO kit to come for transformation ligation and assembly</p>
+mso-bidi-font-family:Arial;color:black'>[71]</span><span style='font-size:16.0pt;
-<p>MC004/7 -all assembled </p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>BH001 -assembled, sequence verify </p>
-<p>BH002 -gblocks not arrived yet</p>
-<p>BH003 -assembled, sequence verify</p>
-<p>BH004 -gblocks not arrived</p>
-<p><b>Zero-Blunt TOPT Ligation and Transformation</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>We will be ligating and transforming four samples in order to allow the bacteria to amplify blocks 1 of MC002 and MC003. </p>
+mso-bidi-font-family:Arial;color:black'>[73]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<ul class=3D"small"><li>MC002_b1</li></ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>MC003_b1</li>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<li>MC002_b1 after PCR</li>
-<li>MC003_b1 after PCR </li></ul>
-<p><blockquote>Ligation</blockquote></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="Picture_x0020_21" o:spid="_x0000_i1045" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/yywO3_6ssAoFt3Ot5zPOiODBmb_5s3XueMAxaDWCXje-NswHlkYVDNdjcJUL0f06nlGmCrdQNWz3DZH6OknRjkQZX2GAjeRTLqxtdbdimMQ0U4Y1dbxEAkOKTHXQafnFhWf0irE"
+ style='width:708.75pt;height:255pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/9/9b/UChicago2015Notebook_filesimage041.png" o:title="yywO3_6ssAoFt3Ot5zPOiODBmb_5s3XueMAxaDWCXje-NswHlkYVDNdjcJUL0f06nlGmCrdQNWz3DZH6OknRjkQZX2GAjeRTLqxtdbdimMQ0U4Y1dbxEAkOKTHXQafnFhWf0irE"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=945 height=340
+src="..//wiki/images/4/4b/UChicago2015Notebook_filesimage042.jpg"
+alt="https://lh6.googleusercontent.com/yywO3_6ssAoFt3Ot5zPOiODBmb_5s3XueMAxaDWCXje-NswHlkYVDNdjcJUL0f06nlGmCrdQNWz3DZH6OknRjkQZX2GAjeRTLqxtdbdimMQ0U4Y1dbxEAkOKTHXQafnFhWf0irE"
+v:shapes="Picture_x0020_21"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<ol><li>Combine 2uL product, 1 uL provided salt solution from pTOPO kit, 2uL H20, 1 uL pCR II-Blunt-TOPO vector </li>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Pipette up and down a few times to mix. </li>
+mso-bidi-font-family:Arial;color:black'>^Things sent.</span><span
-<li>Incubate 5 min at room temperature.   </li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>No overnight incubation needed!  But you can leave it overnight at 4=C2=B0C if you cannot proceed with the transformation right away, </li></ol>
-<p><blockquote>Transformation</blockquote></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<ol><li>Thaw one vial of Chemically Competent E. coli cells per transformation on ice. </li>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Add 2=CE=BCl ligation product to the thawed cells.  Keep on ice for 30 mins. </li>
+mso-bidi-font-family:Arial;color:black'>Transformation of BH001:</span></b><span
-<li>Transfer vials to a 42=C2=B0C water bath for 45 seconds. Immediately return the tubes to ice for 2 min. </li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Add 250=CE=BCl room temperature SOC orLB to each vial.   </li>
-<li>Incubate for 1 hr at 37=C2=B0C. </li>
-<li>Plate 200=CE=BCl cells on LB + kanamycin plates. </li>
-<li>Incubate overnight at 37=C2=B0C</li></ol>
-<p>  *The vector confers resistance to kanamycin, NOT ampicillin.  </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>DNA was transformed into competent
+cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 <span
+class=SpellE>transformants</span>/ng (RbCl2) </u></span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p> pTOPO kit) </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p> <i>  Note: if you do not have a sufficient number of tranformants in 200=CE=BCl, repeat the transformation, do a short spin (20=E2=80=9030 sec) to gently pellet your cells just before plating.  Remove 100=CE=BCl of the supernatant, resuspend the cells by pipeting up and down.</i></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo29;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Remove cells from freezer, incubate
+tubes on ice <o:p></o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo30;
-<p>Set up PCR for amplifying MC002_b1 and MC003_b1. The products from this amplification (correct size ~1850-1970 bps) will be ligated and transformed using the TOPO zero blunt kit in order to have more specific inserts. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<p>	Set up 2 50uL 1X PCR Reactions:</p>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
-<ul class=3D"small"><li>dNTPS: 1uL</li></ul>
+Garamond;color:black'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<li>H2O: 31 uL</li>
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>DNAP: 0.5 uL</li>
+mso-bidi-font-family:Arial;color:black'>Add 1 <span class=SpellE>uL</span> DNA
-<li>DNA: 1 uL (gblocks MC002_b1 and MC003_b1)</li>
+(50 <span class=SpellE>pg</span>//<span class=SpellE>uL</span>) into competent
-<li>DMSO: 1.5uL </li>
+cell tube <o:p></o:p></span></p>
-<li>HF Buffer: 10uL</li>
-<li>F Primer: 2.5uL (MC029)</li>
-<li>R Primer: 2.5 uL (MC010 for MC002_b1 and MC014 for MC003_b1)</li></ul>
-<p>Thermocycler settings for gBlock PCR</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo31;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube on ice for 30 mins<o:p></o:p></span></p>
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds </p><p>30 Cycles</p><p>98=C2=B0C</p><p>66=C2=B0C </p><p>72=C2=B0C</p><p>10 seconds</p><p>30 seconds</p><p>1.65 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo32;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube 42C water bath for 1 min
+heat shock<o:p></o:p></span></p>
-<p><b>Innoculation </b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo33;
-<p><b>	</b>Colonies from MC001 plates were inoculated as colony PCR results were ambiguous. Samples: 115,116,117,118,125,126,127,128. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>19.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube ice 5 mins <o:p></o:p></span></p>
-<ol><li>Aliquot enough LB for 2mL/sample into a Falcon tube. Pipette 2mL of LB into each culture tube/sample. </li>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo34;
-<li>Pipette all of the colony suspension into the culture tube as well. </li>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<li>Put in antibiotic to act as primary screening for insert -Cam 1uL, Amp 4uL (antibiotics stored in the -20 fridge) </li>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
-<li>Place culture tubes into shaker at 37C overnight (~16 hrs) </li></ol>
+Garamond;color:black'><span style='mso-list:Ignore'>20.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Rescue cells by pipetting 900 <span
+class=SpellE>uL</span> LB into tube (use sterile flame)<o:p></o:p></span></p>
-<p><b>Gel Electrophoresis </b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo35;
-<p><b>	</b>1% agarose gel used to run gel electrophoresis for MC002_b1 and MC003_b1. Gel run under 120V for 30 mins. Gel extraction of estimated product size conducted under blue light. 5uL 1kB Plus Invitrogen ladder loaded. 50uL of samples loaded. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<p><b>Image</b></p>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>21.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube in shaker at 37C/1100 RPM
+for 1 hour<o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo36;
-[[File:UChicagoGel21.png]]
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>22.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Heat Cam plate in incubator as cold
+plate reduces <span class=SpellE>efficiency,complete</span> while cells shaking<o:p></o:p></span></p>
-<p><b>8/19/15</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo37;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>23.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Collect pellet, spin 3000 <span
+class=SpellE>rcf</span>/<span class=SpellE>gs</span> for 3 mins <o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo38;
-[[File:UChicagoPlate8.png]]
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>24.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decant 800 <span class=SpellE>uL</span>
+of supernatant <o:p></o:p></span></p>
-<p><b>Transformation Results</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo39;
-<p><b>	</b>Bacterial lawns seen on all plates. Perhaps issue with plates or competent cells are naturally resistant to kanamycin? Will try dilutions if cells are very efficient as cells could also be very conducive to transformation. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>25.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use glass beads (5-6) per section (use
+sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo40;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>26.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Mix pellet, pipetted 200 <span
+class=SpellE>uL</span> in total<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo41;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>27.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Shake with beads and remove <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l14 level1 lfo42;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>28.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate plate overnight 37C<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/13/15-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Sequencing Results</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>Analyzed samples 124 and 121 using Blast and chromatograms. Both samples did not have whole insert -MC031 gave no results for both samples. In both samples sequencing only at end of insert (part of KaiC w/KaiB and suffix) resulted.</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Miniprep-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>Miniprep inoculations 115,116,117,118,125,126,127,128.</p>
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
-<ol><li>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all medium. </li>
+ id="Picture_x0020_22" o:spid="_x0000_i1044" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/5PRzIarnf4BvFtVUcaOaWS7d_qAewOFHKFvJJvYD4k0uhJCmJEIx757jQ9ru2wKIPW02xwaAK5v_T_oUFl67dbZzkFNBHazN1w9bkjpt1hSfn1Qw_wFuolAG096Kxc7F0k0ZQPA"
-<li>Resuspend cells by adding 250uL of resuspension buffer R3 with RNase A. Mix up and down until homogenous. </li>
+ style='width:1057.5pt;height:369.75pt;visibility:visible;mso-wrap-style:square'>
-<li>Add 250uL of Lysis buffer (L7) to lyse cells. Mix gently by inverting capped tube.Do not vortex, incubate at room temp for 5 mins. </li>
+ <v:imagedata src="..//wiki/images/c/c9/UChicago2015Notebook_filesimage043.png" o:title="5PRzIarnf4BvFtVUcaOaWS7d_qAewOFHKFvJJvYD4k0uhJCmJEIx757jQ9ru2wKIPW02xwaAK5v_T_oUFl67dbZzkFNBHazN1w9bkjpt1hSfn1Qw_wFuolAG096Kxc7F0k0ZQPA"/>
-<li>Add 350uL of precipitation buffer N4. mix immediately by inverting tube or vigorously shaking. Do not vortex, centrifuge at 20,000 rcfs for 10 mins. </li>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1410 height=493
-<li>Load supernatant (750 uL) into Spin column (machery nagel used) and collection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow through and place column back in wash tube. </li>
+src="..//wiki/images/3/3f/UChicago2015Notebook_filesimage044.png"
-<li>Add 500 uL of wash buffer W10 with ethanol. Incubate at room temp for 1 min, centrifuge column at 12,000gs for 1 min. Discard flow through. <strong><em>(*Optional Wash step)</em></strong></li>
+alt="https://lh3.googleusercontent.com/5PRzIarnf4BvFtVUcaOaWS7d_qAewOFHKFvJJvYD4k0uhJCmJEIx757jQ9ru2wKIPW02xwaAK5v_T_oUFl67dbZzkFNBHazN1w9bkjpt1hSfn1Qw_wFuolAG096Kxc7F0k0ZQPA"
-<li>Add 700 uL of wash buffer W9 with ethanol. centrifuge column at 12,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs. Discard flow through. </li>
+v:shapes="Picture_x0020_22"><![endif]></span><span style='font-size:16.0pt;
-<li>Place spin column into microfuge tube. Add 50uL of TE Buffer to center of column. Incubate column in room temp for 1 min. </li>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop DNA. Store at 4C short term, -20C long term. </li></ol>
-<p>Nanodrop results-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>115: 91.8 ng/uL, 260/280: 1.90</p>
+mso-bidi-font-family:Arial;color:black'>Transformation Efficiency of BH001-</span><span
-<p>116: 172.0 ng/uL, 260/280: 1.82</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>117: 152.0 ng/uL, 260/280: 1.91</p>
-<p>118: 120.4 ng/uL, 260/280: 1.81</p>
-<p>125: 187.0 ng/uL, 260/280: 1.88</p>
-<p>126: 149.2 ng/uL, 260/280: 1.88</p>
-<p>127: 211.0 ng/uL, 260/280: 1.87</p>
-<p>128: 222.8 ng/uL, 260/280: 1.89</p>
+<p class=MsoNormal><span class=SpellE><span class=GramE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>amt</span></span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> <span class=SpellE>dna</span> used/plate</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>PCR </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>Set up PCR for both miniprep PCR and PCR of MC002_b1 and MC003_b1 to redo ligation and transformation. </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Set up 2 50uL 1X PCR Reactions for MC002_b1 and MC003_b1:</p>
-<li>dNTPS: 1uL</li>
-<li>H2O: 31 uL</li>
-<li>DNAP: 0.5 uL</li>
-<li>DNA: 1 uL (gblocks MC002_b1 and MC003_b1)</li>
-<li>DMSO: 1.5uL </li>
-<li>HF Buffer: 10uL</li>
-<li>F Primer: 2.5uL (MC029)</li>
-<li>R Primer: 2.5 uL (MC010 for MC002_b1 and MC014 for MC003_b1)</li></ul>
-<p>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2 uL DNA):</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	HF Buffer: 18.00</p>
+mso-bidi-font-family:Arial;color:black'>Plates 1 and 2 were discarded, a colony
-<p>	DMSO: 2.70 uL</p>
+PCR was done on 6 samples from Plate 3</span><span style='font-size:16.0pt;
-<p>	dNTPS: 1.80</p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	H2O: 55.</p>
-<p>	DNAP: 4.25 uL</p>
-<p>	Used 4.50 uL of 10X MC003 and MC004 for mix. </p>
-<p>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds </p><p>30 Cycles</p><p>98=C2=B0C</p><p>67=C2=B0C </p><p>72=C2=B0C</p><p>10 seconds</p><p>30 seconds</p><p>1:40 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>1% agarose gel run at 120V for 30 mins. Loaded 10uL and 45 uL for minipreps and MC002/3 gel extractions respectively. Loaded 5uL of 1kB Plus Invitrogen ladder. </p>
+mso-bidi-font-family:Arial;color:black'>They were <span class=SpellE>PCRed</span>
+separately with two different annealing temperatures, 61.6 and 66. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Image- </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoLadder2.png]]
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly</span></b><span
-<html>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/20</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson assembly using 5uL of <span
+class=SpellE><span class=GramE>gibson</span></span> master mix 2x conducted.
+Assembled on ice. Incubated for 1 hour 50C <span class=SpellE>heatblock</span>.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Sequences Submitted</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>MC001 samples submitted in morning for sequencing. 4uM primer stock made by diluting in water (1:24 primer:water ratio). 10 uL of miniprepped DNA submitted with 10uL of 4uM primers. Samples 115,117, 125, 127, 128 sent. </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_23"
+ o:spid="_x0000_i1043" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/8zTuzPyZXuqC49j0qL97Utg1_Tn4eW4vcWzlUn88_w52ynrlGdEhfdH4ltnNsUx6ChIfyBv7dr7mchO3S7soqYQtHo1AwHb3YXi4ZffN2UxsdrgkJSbi9oK9tVMzr20TsLL3YF0"
+ style='width:1020.75pt;height:60.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/1/16/UChicago2015Notebook_filesimage045.png" o:title="8zTuzPyZXuqC49j0qL97Utg1_Tn4eW4vcWzlUn88_w52ynrlGdEhfdH4ltnNsUx6ChIfyBv7dr7mchO3S7soqYQtHo1AwHb3YXi4ZffN2UxsdrgkJSbi9oK9tVMzr20TsLL3YF0"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1361 height=81
+src="..//wiki/images/e/e0/UChicago2015Notebook_filesimage046.png"
+alt="https://lh5.googleusercontent.com/8zTuzPyZXuqC49j0qL97Utg1_Tn4eW4vcWzlUn88_w52ynrlGdEhfdH4ltnNsUx6ChIfyBv7dr7mchO3S7soqYQtHo1AwHb3YXi4ZffN2UxsdrgkJSbi9oK9tVMzr20TsLL3YF0"
+v:shapes="Picture_x0020_23"><![endif]></span><o:p></o:p></span></p>
-<p>Primers used: </p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>VF2 (1)</p>
+mso-bidi-font-family:Arial;color:black'>PCR-</span></b><span style='font-size:
-<p>MC003 (2)</p>
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>MC031 (3)</p>
-<p>MC032 (4)</p>
-<p>MC006 (5)</p>
-<p>MC033 (6)</p>
-<p>MC008 (7)</p>
-<p>VR (8)  </p>
-<p><b>Gibson Assembly</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>New primers for MC001 arrived. Proceeded with Gibson assesmbly. Incubated mix at 50C for 1 hour.</p>
+mso-bidi-font-family:Arial;color:black'>Prepared 2 50uL 1X PCR samples. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-[[File:UChicagoTable9.png]]
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
-<html>
+style='mso-tab-count:1'>          </span>HF Buffer -10 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>Set up PCR for MC002_b1, MC003_b1, MC001_b1, MC001_b2, and MC001 Gibson products. Total 7 reaction (MC002/3 block 1s sampled twice as one will be used for gel extraction then TOPO ligation/transformation, the other will be used to amplify a second time). </p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DMSO -1.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Phusion 7.5 x of 1X mix</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>	HF Buffer -75.00 uL</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
-<p>	DMSO -11.25 uL</p>
+style='mso-tab-count:1'>          </span>DNTPs -1 <span class=SpellE>uL</span> </span><span
-<p>	DNTPs -7.50 uL </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	H2O -232.50 uL</p>
-<p>	Phusion DNAP -3.75 uL</p>
-<p>Add 44uL of PCR Mix to 2.5 of F and R Primer each and 1 uL of DNA</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>H2O -31 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>DNA : Primers</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>MC002_b1 : MC029, MC010</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
-<p>MC003_b1 : MC029, MC014</p>
+style='mso-tab-count:1'>          </span><span class=SpellE>Phusion</span> DNAP
-<p>Gibson rxn : 1b1F, 1b2R</p>
+-0.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
-<p>MC001_b1 : 1b1F, 1b1R</p>
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>MC002_b2:  1b2F, 1b2R </p>
-<p>Thermocycler settings:</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>F Primer 10 <span class=SpellE>uM</span>
+MC003 -2.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds </p><p>30 Cycles</p><p>98=C2=B0C</p><p>66=C2=B0C </p><p>72=C2=B0C</p><p>10 seconds</p><p>30 seconds</p><p>1:30 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>R Primer 10uM MC004 -2.5 <span
+class=SpellE>uL</span> </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrohporesis-</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p><b>	</b>1% Agarose gel run at 120V for 30 mins. 45 uL of samples loaded. Hard to see bands under blue light. Used UV light for quick clarification of size. No products from MC001 Gibson seen. MC001_b1 and MC001_b2 extracted separately. </p>
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DNA (Gibson assembly <span
+class=SpellE>rxn</span> 8/13) -1uL </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/21</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>Due to failure of yesterday=E2=80=99s PCR, gradient PCR was set up in order to determine optimal anneal temperature. </p>
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   Phusion 5X HF Buffer =3D 15 uL</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>o   dNTPs 10 mM  =3D 1.5  uL</p>
-<p>o   Phusion DNAP =3D 0.75 uL</p>
-<p>o   H2O =3D 46.5 uL</p>
-<p>o   DMSO =3D 2.25 uL</p>
-<p>o   DNA =3D1.5 uL</p>
-<p>o   F Primer MC003=3D 3.75 uL</p>
-<p>o   R Primer MC004 =3D3.75 uL</p>
-<p>10uL Master mix aliquoted into 7 samples.</p>
-<p>Thermocycler settings- 98o 2 min, 98o 15 s, 60o, 62o, 64o, 66o, 68o, 70o, 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72.0o</p>
-<p>T=3D66.0o, G=3D6.0o for 30 cycles</p>
-<p><b>Gel Purification </b></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-<p><b>	</b>MC001_b1 and MC001_b2 extracted yesterday were purified. Used Machery-Nagel protocol. </p>
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>65°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 <span
+  class=SpellE>secondsc</span></span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1.65 mins (30s x 3.3kb -largest product)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-<p>Weights-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>1b1 -13.4 mg</p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>1b2 - 133.4 mg</p>
-<p>Nanodrop results-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>1b1 - 13.1 ng/uL</p>
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings for Mini-Prep
-<p>1b2 -19.2 ng/ul</p>
+PCR- Low Temp</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gel Electrophoresis</b></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-<p><b>	</b>1% agarose gel run for gradient PCR. 120V for 30 mins. 1kB Invitrogen ladder used. 10uL of sample loaded. Expected product size ~3000 bps. No expected product size appeared on gel. Perhaps indicates issues with Gibson assembly.  24 inoculated colony PCRs also run on separate gel. 3 colonies chosen for inoculation and miniprep: 3,9 and 22. </p>
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>120 seconds</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>61.6°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>(NEB - DMSO%*0.8)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>15 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1 min (30s x 1.8kb -largest product)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>5 min</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-<p><b>Image-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoGel3.png]]
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings for Mini-Prep
-[[File:UChicagoGel4.png]]
+PCR- High Temp</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<html>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Gibson- </b></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-<p><b>	</b>Gibson of MC001 repeated. Incubated mix at 50C for 1 hour. </p>
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>120 seconds</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>66°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>15 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1 min (30s x 1.8kb -largest product)</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>5 min</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoTable11.png]]
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<html>
-<p><b>PCR- </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>PCR set up for MC001 blocks and MC001 8/21 Gibson. 6 samples in total. 1X PCR Mix for each sample set up:</p>
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
-<p>	DMAP: 0.5 uL</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>	DMSO: 1.5 uL</p>
-<p>	dNTPS: 1 uL</p>
-<p>	DNA: 1uL</p>
-<p>	HF Buffer: 10 uL</p>
-<p>	H2O: 31 uL</p>
-<p>	F Primer: 2.5 uL (10uM)</p>
-<p>	R Primer: 2.5 uL (10uM) </p>
-<p>DNA (Primers): MC001_b1 (MC001_b1_F_new, MC001_b1_R_new), MC001_b1 (MC001_b2_F_new, MC001_b2_R_new), MC001 Gibson (MC001_b1_F_new, MC001_b2_R_new)</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>100 mL of 1% agarose gel run 120V, 30 mins. Samples loaded include
+<span class=SpellE>uL</span> of BH003 <span class=SpellE>biobrick</span> and
+<span class=SpellE>uL</span> of colony PCRs. 5 <span class=SpellE>uL</span>
+kB plus Invitrogen ladder loaded. Colony PCRs yielded no results, <span
+class=SpellE>biobrick</span> BH003 extracted using gel punches (seen in image).
+First column of BH3 seemed to give very low yield (light band not strong band
+seen before extraction).</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>PCR Conducted one at high anneal temperature (68C) one at low anneal temperature (64C)</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and other for 50uL samples)-</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denaturation</p><p>98=C2=B0C</p><p>30 seconds </p><p>30 Cycles</p><p>98=C2=B0C</p><p>68 or 64=C2=B0C </p><p>72=C2=B0C</p><p>10 seconds</p><p>30 seconds</p><p>1:30 mins</p><p>Final Extension</p><p>72=C2=B0C</p><p>10 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+mso-bidi-font-family:Arial;color:black'>Image-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_24" o:spid="_x0000_i1042"
+ type="#_x0000_t75" alt="https://lh4.googleusercontent.com/lRcesA15fcD4p7N0gVbO38n1_qCfUH__rpM3Jlq_Cw8EiV0VMSIYJEWtSSOCaWaNFRKzBFepZ83Oimx-tdts1d1KlrFtgfMg3TDQqVAGctX3ECHvYCVV0lGdoEujvH254yCRZF0"
+ style='width:627.75pt;height:519pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/5/58/UChicago2015Notebook_filesimage047.png" o:title="lRcesA15fcD4p7N0gVbO38n1_qCfUH__rpM3Jlq_Cw8EiV0VMSIYJEWtSSOCaWaNFRKzBFepZ83Oimx-tdts1d1KlrFtgfMg3TDQqVAGctX3ECHvYCVV0lGdoEujvH254yCRZF0"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=837 height=692
+src="..//wiki/images/c/c2/UChicago2015Notebook_filesimage048.png"
+alt="https://lh4.googleusercontent.com/lRcesA15fcD4p7N0gVbO38n1_qCfUH__rpM3Jlq_Cw8EiV0VMSIYJEWtSSOCaWaNFRKzBFepZ83Oimx-tdts1d1KlrFtgfMg3TDQqVAGctX3ECHvYCVV0lGdoEujvH254yCRZF0"
+v:shapes="Picture_x0020_24"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>	</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p><b>Gel Electrophoresis-</b></p>
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
-<p><b>	</b>1% agarose gel run at 120V for 30 mins. 1kB Plus invitrogen ladder used. 50uL of samples loaded. Results very inconclusive. </p>
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
-<p><b>Image-</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Extraction-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/24</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH003 <span class=SpellE>biobrick</span>
+extracted from gel. <span class=SpellE>Machery</span>-Nagel protocol used for
+gel extraction. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Miniprep-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	Inoculated TOPO MC002_b1 and MC003_b1 samples miniprepped using invitrogen protocol. </p>
+mso-bidi-font-family:Arial;color:black'>Weight of gel: 85.2 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Miniprep inoculations 115,116,117,118,125,126,127,128.</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<ol start="10">
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<li>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all medium. </li>
-<li>Resuspend cells by adding 250uL of resuspension buffer R3 with RNase A. Mix up and down until homogenous. </li>
-<li>Add 250uL of Lysis buffer (L7) to lyse cells. Mix gently by inverting capped tube.Do not vortex, incubate at room temp for 5 mins. </li>
-<li>Add 350uL of precipitation buffer N4. mix immediately by inverting tube or vigorously shaking. Do not vortex, centrifuge at 20,000 rcfs for 10 mins. </li>
-<li>Load supernatant (750 uL) into Spin column (machery nagel used) and collection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow through and place column back in wash tube. </li>
-<li>Add 500 uL of wash buffer W10 with ethanol. Incubate at room temp for 1 min, centrifuge column at 12,000gs for 1 min. Discard flow through. <strong><em>(*Optional Wash step)</em></strong></li>
-<li>Add 700 uL of wash buffer W9 with ethanol. centrifuge column at 12,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs. Discard flow through. </li>
-<li>Place spin column into microfuge tube. Add 50uL of TE Buffer to center of column. Incubate column in room temp for 1 min. </li>
-<li>Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop DNA. Store at 4C short term, -20C long term. </li></ol>
-<p>Nanodrop results-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A21: </p>
+mso-bidi-font-family:Arial;color:black'>Used <span class=SpellE>Machery</span>-Nagel
-<p>A22: </p>
+clean up method:</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A31:</p>
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>A32: </p>
-<p>B21: </p>
-<p>B22: </p>
-<p>B31: </p>
-<p>B32: </p>
-<p>None were sufficient enough to send for sequencing. Likely errors also due to overgrowth of colonies. </p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo43;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 200uL NTI Binding buffer / 100 mg
+gel <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo44;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate at 50C for 10 mins<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo45;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer solution to spin column and
+collection tube. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo46;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p><b>PCR </b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo47;
-<p>Gradient PCR of only MC001 blocks conducted. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Discard flow through. Add 700 <span
+class=SpellE>uL</span> of wash buffer NT3 to spin column.<o:p></o:p></span></p>
-<p>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo48;
-<p>o   Phusion 5X HF Buffer =3D 37.5 uL</p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<p>o   dNTPs 10 mM  =3D 3.75  uL</p>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
-<p>o   Phusion DNAP =3D 1.875 uL</p>
+Garamond;color:black'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p>o   H2O =3D 116.25 uL</p>
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   DMSO =3D 5.625 uL</p>
+mso-bidi-font-family:Arial;color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p>o   DNA =3D3.75 uL</p>
-<p>o   F Primer MC003=3D 9.375 uL</p>
-<p>o   R Primer MC004 =3D9.375 uL</p>
-<p>10uL Master mix aliquoted into 7 samples.</p>
-<p>Thermocycler settings- 98o 2 min, 98o 15 s, 60o, 62o, 64o, 66o, 68o, 70o, 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
-<p>Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72.0o</p>
-<p>T=3D66.0o, G=3D6.0o for 30 cycles</p>
-<p><b>Gel Electrophoresis</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo49;
-<p>1 % agarose gel run for 30 mins at 120V. 25 uL of samples loaded. 1kB plus Invitrogen ladder loaded. Only sufficient product seen for block 2. Indicates issues with block 1. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Discard flow through. Add 700 <span
+class=SpellE>uL</span> of wash buffer NT3 to spin column. <o:p></o:p></span></p>
-<p><b>Image</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo50;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>19.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-</html>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo51;
-[[File:UChicagoGel5.png]]
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<html>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>20.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Spin at 11,000g (RCFs) for 1 min to dry
+silica membrane <o:p></o:p></span></p>
-<p><b>8/25</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo52;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>21.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Elute DNA with 15 <span class=SpellE>uL</span>
+of NE elution buffer. Spin at 11,000g (RCFs) for 30s<o:p></o:p></span></p>
-<p>Sequencing Results: Found a sample (127) containing entire MC001 insert! </p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l9 level1 lfo53;
-<p>Innoculated sample 127 and prepared for induction with L-Rhamnose. </p>
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
-<p>Re-plated TOPO reactions for MC002/3 as overgrowth of colonies on previous plates. </p>
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
-<p>Set up inductions however, cultured at 37C instead of 30C. Pay attention to this, this is important, all the protocols have induced L-Rhamnose at 30C </p>
+Garamond;color:black'><span style='mso-list:Ignore'>22.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span class=SpellE><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> (use EB buffer as blank, load 1.5 <span class=SpellE>uL</span>
+sample)<o:p></o:p></span></p>
-<p><b>8/26</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p>Start over the inductions, follow the igem protocol file for help. You will need to make more LB. I have already made a few glycerol stocks. You will need to set up a 2mL innoculation in a culture tube as well as a 40mL innoculation in a flask. The 40mL innoculation will be used for inductions. [Finished]</p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> purity- 14.4 ng/<span class=SpellE>uL</span>, 260/280- 1.55</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>Redo TOPO transformations. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>8/27</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR-</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/28</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Made 4 1X 50uL samples to PCR ampicillin backbone. Used 1 <span
+class=SpellE>uL</span> of 25ng/<span class=SpellE>uL</span> amp linearized
+backbone. Primers MC001 and MC002. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>8/31</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Reviewed work done over past three days. Discussed ways to resolve western blot issue. Seems to be a lot of non-specific binding -we see blots of many different sizes. Blots of antibodied products are very bright relative to ladder. Kevin mentioned degradation could be an issue (would cause lots of different sizes in blots). On closer examination, 0% rhamnose has no (barely any) protein. Updated notebook, e-mailed Justin and Kevin for trouble shooting. </p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Master Mix Recipe-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 50 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>BH- Type up lysing assay and bradford assay -give some specifics about western blot (how much each sample, and calculations) </p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 5 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p>E-mail sleep people</p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 2.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>9/1</b></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 155 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Induction Solutions</b></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
-<p>In order to re-do induction western blots, made new 10% Rhamnose w/v solution by diluting 1 g of Rhamnose in 9 mL of water to bring final volume to 10mL.</p>
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 7.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Culture</b></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
-<p>Set up two 40 mL cultures of sample 127 in 250 mL flasks. Added 20uL of Cam in each, 40 mL of LB and 10uL of 2 mL sample 127 overnight culture. Note: 2mL overnight culture has been stored in room temperature for past four days -may also have to start from new glycerol stock. 40mL cultures placed in 37C incubator at 11:50. </p>
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
-<p><b>Dilutions of Rhamnose Inducer Samples</b></p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
-<p>As discussed in meetings, errors with previous western blot likely to be caused due to overexpression of pRhamnose. iGEM team used low copy number plasmid. Re-made inducer solutions using 1/10 of % from 0.001 - 0.1% in order to test lower gradient. Induction started at 2:30 am when OD600 of 40 mL innoculations was 1.6(?) </p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA (products from
+Gibson 8/4)= 1.0uL</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>9/2</b></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
-<p><b>Minimal Media</b></p>
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
-<p>Ordered M9 minimal media 5X salts on Rust Lab tab. Should take till friday to arrive. </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC001= 2.5uL</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Restriction Digest </b></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
-<p>Protocol from iGEM.</p>
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
-<h2>Digest</h2>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
-<ul class=3D"small"><li>Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)</li></ul>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<ul class=3D"small"><li>5 ul NEB Buffer 2 (use CutSmart)</li></ul>
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC002 =2.55uL</span><span
-<li>0.5 ul BSA</li>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<li>0.5 ul EcoRI-HF</li>
-<li>0.5 ul PstI</li>
-<li>0.5 ul DpnI (Used to digest any template DNA from production)</li>
-<li>18 ul dH20</li>
-<ul class=3D"small"><li>Digest Plasmid Backbone</li></ul>
-<ul class=3D"small"><li>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)</li></ul>
-<li>Add 4 ul of Enzyme Master Mix</li>
-<li>Digest 37C/30 min, heat kill 80C/20 min</li></ul>
-<h2>Ligation</h2>
-<ul class=3D"small"><li>Add 2ul of digested plasmid backbone (25 ng)</li></ul>
-<li>Add 1 ul T4 DNA ligase buffer. <b>Note:</b> Do not use quick ligase</li>
-<li>Add 0.5 ul T4 DNA ligase</li>
-<li>Add water to 10 ul</li>
-<li>Ligate 16C/30 min, heat kill 80C/20 min</li>
-<li>Transform with 1-2 ul of product</li></ul>
-<p><b>Restreaked TOPO plate </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Transformed new TOPO reactions for MC02/3 and BH002/3</b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>Set up Gibson of BH002/3</b></p>
-<p><b>9/3 </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Lysed cells after induction </b></p>
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings<span class=GramE>:98C</span>
-<p><b>Bradford Assay </b></p>
+s, 25 cycles: 98C 10s, 69C 30s, 72C 1.5m, 72.0C 10 min, 4C hold</span><span
-<p><b>Conducted Western blot </b></p>
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>Ran colony PCR of TOPO reactions -only MC003 TOPO worked. BH002/3 Gibsons worked, no TOPO results  for MC002, BH002, BH003. </b></p>
-<p><b>Inoculated negative control. </b></p>
-<p><b>9/4/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Set up Rhamnose time course experiment and rhamnose gradient experiment. Both used M9 media and LB media. M9 media arrived. </b></p>
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
-<p><b>9/7/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Miniprepped colony PCR for MC003, BH002, BH003. </b></p>
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>9/8/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Set up bradford of time-course and rhamnose gradient. Ran gel and transfer of assays. Ran negative vector control this time. </b></p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Given success of <span class=SpellE><span class=GramE>gibson</span></span>
+and transformation with BH001, decided to try assemble pMC001 and transform
+directly. Reaction incubated on <span class=SpellE>heatblock</span> for 1 hour
+at 50C. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>9/9</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Finished western blot, imaged gel. Blotted only for KaiA and KaiC. </b></p>
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="Picture_x0020_25" o:spid="_x0000_i1041" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/xOvXy9DorVztTDX1w6AnFASvTD8PH2Hxa0ggOORWpHMbMA04L100JIENqG2Rvbo92iDW8wUc5VBZyhZCIeJwZa5SJ39jFKIf22tD4olxWyVXL2dTdTNv-AipORA8bQTHMlt3Oi8"
+ style='width:1020.75pt;height:81pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/8/8f/UChicago2015Notebook_filesimage049.png" o:title="xOvXy9DorVztTDX1w6AnFASvTD8PH2Hxa0ggOORWpHMbMA04L100JIENqG2Rvbo92iDW8wUc5VBZyhZCIeJwZa5SJ39jFKIf22tD4olxWyVXL2dTdTNv-AipORA8bQTHMlt3Oi8"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1361 height=108
+src="..//wiki/images/7/72/UChicago2015Notebook_filesimage050.png"
+alt="https://lh5.googleusercontent.com/xOvXy9DorVztTDX1w6AnFASvTD8PH2Hxa0ggOORWpHMbMA04L100JIENqG2Rvbo92iDW8wUc5VBZyhZCIeJwZa5SJ39jFKIf22tD4olxWyVXL2dTdTNv-AipORA8bQTHMlt3Oi8"
+v:shapes="Picture_x0020_25"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<p><b>9/10</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Set up induction again, be careful of transfer where errors seem to occur. Blot for Kai B from previous assay. </b></p>
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
-<!-- autogen end -->
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>50 mL of 1% agarose gel made to run amp backbone amplifications.
+.5 <span class=SpellE>uL</span> EtBr used. Gel run 120V for 30 mins. 1 kB Plus
+Invitrogen ladder used. 45 <span class=SpellE>uL</span> of samples loaded. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image- </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_26" o:spid="_x0000_i1040"
+ type="#_x0000_t75" alt="https://lh4.googleusercontent.com/-7lj8c8QrXxqZRW7eACtWpuPUxMEmKh238gYNwhIUyD6E4SVnF6Eq5fM_3CvDe-I4ANqe8yK11jmws019qwa9FNVUapLFRZF0LXuz5yN-uY6cmN7kk73d-XZG_FvyoVKKvXe3UU"
+ style='width:550.5pt;height:347.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/e/ec/UChicago2015Notebook_filesimage051.png" o:title="-7lj8c8QrXxqZRW7eACtWpuPUxMEmKh238gYNwhIUyD6E4SVnF6Eq5fM_3CvDe-I4ANqe8yK11jmws019qwa9FNVUapLFRZF0LXuz5yN-uY6cmN7kk73d-XZG_FvyoVKKvXe3UU"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=734 height=463
+src="..//wiki/images/1/15/UChicago2015Notebook_filesimage052.png"
+alt="https://lh4.googleusercontent.com/-7lj8c8QrXxqZRW7eACtWpuPUxMEmKh238gYNwhIUyD6E4SVnF6Eq5fM_3CvDe-I4ANqe8yK11jmws019qwa9FNVUapLFRZF0LXuz5yN-uY6cmN7kk73d-XZG_FvyoVKKvXe3UU"
+v:shapes="Picture_x0020_26"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Extraction and Purification-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ampicillin <span class=GramE>backbone
+&nbsp;extracted</span> from gel. <span class=SpellE>Machery</span>-Nagel
+protocol used for gel extraction. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Weight of gel samples:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A1 -135.8 mg (271.6 <span class=SpellE>uL</span>
+of binding buffer NTI used)</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A2 -201.4mg (402.8 <span class=SpellE>uL</span>
+of binding buffer NTI used) </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> concentrations of ampicillin backbone samples</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A1 - 32.3 ng/<span class=SpellE>uL</span>,
+/280- 1.84</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A2 - 33.4 ng/<span class=SpellE>uL</span>,
+/280- 1.59 </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson assembly of ampicillin backbone
+to BH003 insert conducted. Used 5uL of <span class=SpellE><span class=GramE>gibson</span></span>
+master mix 2x conducted. Assembled on ice. Incubated for 1 hour 50C <span
+class=SpellE>heatblock</span>.</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="Picture_x0020_27" o:spid="_x0000_i1039" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/dufpeo0rHYirlXsq8VIsPbQnu8-K0nIHzB72pqqGtEr_H79ENqE4asCGd9FfDE-xVA9PzD89aB0VHN-MB_az-0qyPpR6Nz0VhZIhpn1t_8dhemST0QvAYAyIRvs7lVE7kLw1V4U"
+ style='width:1020.75pt;height:60.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/6/6e/UChicago2015Notebook_filesimage053.png" o:title="dufpeo0rHYirlXsq8VIsPbQnu8-K0nIHzB72pqqGtEr_H79ENqE4asCGd9FfDE-xVA9PzD89aB0VHN-MB_az-0qyPpR6Nz0VhZIhpn1t_8dhemST0QvAYAyIRvs7lVE7kLw1V4U"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1361 height=81
+src="..//wiki/images/b/bf/UChicago2015Notebook_filesimage054.png"
+alt="https://lh6.googleusercontent.com/dufpeo0rHYirlXsq8VIsPbQnu8-K0nIHzB72pqqGtEr_H79ENqE4asCGd9FfDE-xVA9PzD89aB0VHN-MB_az-0qyPpR6Nz0VhZIhpn1t_8dhemST0QvAYAyIRvs7lVE7kLw1V4U"
+v:shapes="Picture_x0020_27"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation of BH003:</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>DNA was transformed into competent
+cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 <span
+class=SpellE>transformants</span>/ng (RbCl2) </u></span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo54;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>29.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Remove cells from freezer, incubate
+tubes on ice <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo55;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>30.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 1 <span class=SpellE>uL</span> DNA
+(50 <span class=SpellE>pg</span>//<span class=SpellE>uL</span>) into competent
+cell tube <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo56;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>31.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube on ice for 30 mins<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo57;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>32.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube 42C water bath for 1 min
+heat shock<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo58;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>33.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube ice 5 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo59;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>34.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Rescue cells by pipetting 900 <span
+class=SpellE>uL</span> LB into tube (use sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo60;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>35.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate tube in shaker at 37C/1100 RPM
+for 1 hour<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo61;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>36.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Heat Cam plate in incubator as cold
+plate reduces <span class=SpellE>efficiency,complete</span> while cells shaking<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo62;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>37.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Collect pellet, spin 3000 <span
+class=SpellE>rcf</span>/<span class=SpellE>gs</span> for 3 mins <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo63;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>38.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Decant 800 <span class=SpellE>uL</span>
+of supernatant <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo64;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>39.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Use glass beads (5-6) per section (use
+sterile flame)<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo65;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>40.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Mix pellet, pipetted 200 <span
+class=SpellE>uL</span> in total<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo66;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>41.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Shake with beads and remove <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l0 level1 lfo67;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>42.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Incubate plate overnight 37C<o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/14/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>MC001
+and BH003 gave good results from transformation. Colonies were small, but
+evenly distributed. Colonies could be small because plates incubated late last
+night. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_28"
+ o:spid="_x0000_i1038" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/5JUbotNvkXgOg9G28elqEXo8vw_hgB6DVsmd1B5eBCtc_X_ViBFGAK6zdzKEYGeQetzG8beXSLk5M5Bni2NjK0FqoG52PhI4ZQ_MG2Tx1Y5-UnGtexg2k_zBNkG2owT9R34hbAk"
+ style='width:483.75pt;height:405pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/a/ae/UChicago2015Notebook_filesimage055.png" o:title="5JUbotNvkXgOg9G28elqEXo8vw_hgB6DVsmd1B5eBCtc_X_ViBFGAK6zdzKEYGeQetzG8beXSLk5M5Bni2NjK0FqoG52PhI4ZQ_MG2Tx1Y5-UnGtexg2k_zBNkG2owT9R34hbAk"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=645 height=540
+src="..//wiki/images/5/52/UChicago2015Notebook_filesimage056.png"
+alt="https://lh6.googleusercontent.com/5JUbotNvkXgOg9G28elqEXo8vw_hgB6DVsmd1B5eBCtc_X_ViBFGAK6zdzKEYGeQetzG8beXSLk5M5Bni2NjK0FqoG52PhI4ZQ_MG2Tx1Y5-UnGtexg2k_zBNkG2owT9R34hbAk"
+v:shapes="Picture_x0020_28"><![endif]></span><br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Colony PCR of BH003/MC001-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Diluted one colony from plates with
+most growth into 50uL of dH2O to serve as DNA template. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 8.5 x of 1X PCR Master Mix:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>HF
+Buffer: 85 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DMSO:
+.75 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span><span
+class=SpellE><span class=GramE>dNTPS</span></span>: 8.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>H2O:
+.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DNAP:
+.25 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Used
+.5uL of VF2 and VR each and 1 <span class=SpellE>uL</span> of sample DNA. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings for Colony </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 minutes </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>66°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>15 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>2.5 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequencing Primers for MC001- MC003<span
+class=GramE>,MC031,MC032,MC006,MC033</span>, MC008</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/15/</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>1% Agarose gel run for colony PCR. 1 kB Invitrogen plus ladder
+used. 50 <span class=SpellE>uL</span> of sample loaded. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_29"
+ o:spid="_x0000_i1037" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/emrPKYXBDaFV7qpMaymBc932dySXKmseC0tK9A81xnk90xR-KgwSBn9pA4Jt4xjwzp5o5dAHfnua_M_vHcM91nLUKxyCs18p3_henMI20P8fCj3vD1T3gWxxBikJIG-NYA07plo"
+ style='width:573pt;height:444pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/41/UChicago2015Notebook_filesimage057.png" o:title="emrPKYXBDaFV7qpMaymBc932dySXKmseC0tK9A81xnk90xR-KgwSBn9pA4Jt4xjwzp5o5dAHfnua_M_vHcM91nLUKxyCs18p3_henMI20P8fCj3vD1T3gWxxBikJIG-NYA07plo"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=764 height=592
+src="..//wiki/images/5/55/UChicago2015Notebook_filesimage058.png"
+alt="https://lh3.googleusercontent.com/emrPKYXBDaFV7qpMaymBc932dySXKmseC0tK9A81xnk90xR-KgwSBn9pA4Jt4xjwzp5o5dAHfnua_M_vHcM91nLUKxyCs18p3_henMI20P8fCj3vD1T3gWxxBikJIG-NYA07plo"
+v:shapes="Picture_x0020_29"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/17</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequences Submitted</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC001 note submitted on weekend for
+sequencing. MC001 submitted in morning for sequencing. 4uM primer stock made by
+diluting in water (1:24 <span class=SpellE>primer<span class=GramE>:water</span></span>
+ratio). 14 <span class=SpellE>uL</span> of <span class=SpellE>miniprepped</span>
+DNA submitted with 10uL of 4uM primers. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers used: </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>VF2 (1)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>VR (2)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC003 (3)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC031 (4)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC032 (5)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC006 (6)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC033 (7)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC008 (8) </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Colony PCR</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Colony PCR of MC001 set up again as
+upon closer examination, insert seems too small to be correct. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Diluted one colony from plates with
+most growth into 50uL of dH2O to serve as DNA template. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 9/5x of 1X PCR Master Mix for 10uL
+samples (used 9.8 <span class=SpellE>uL</span> Mix and 0.2 <span class=SpellE>uL</span>
+DNA):</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>HF
+Buffer: 18.00</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DMSO:
+.70 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span><span
+class=SpellE><span class=GramE>dNTPS</span></span>: 1.80</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>H2O:
+.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DNAP:
+.25 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Used
+.50 <span class=SpellE>uL</span> of 10X MC003 and MC004 for mix. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings for Colony </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 minutes </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>66°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>15 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>2.5 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Talked with Justin about ordering
+materials -TOPO kit should come tomorrow. </span></i><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/18</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>0.8% (to separate out larger fragments
+with more efficiency) agarose gel cast. Gel run on 120V for 30 mins. 1kB plus
+Invitrogen ladder used. No results from colony PCR, indicating something wrong
+during PCR. Likely issue with primers? ¾ Used instead of VF2 and VR. Have
+double checked primers are correct -could be issue with insert and primers.
+Note -issues with evaporation in thermocycler, which is why samples
+,116,117,118,128 could not be loaded. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_30"
+ o:spid="_x0000_i1036" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/r6zsedRwioKWtxu0tkFeTi7D_42tqiJY0mG8k6qj8E7kMkYtRZW0w5TzpNGh4g2ICpc5CisNFak7tRlvsuaNWZSWdBa8hctZ45hCaNhC9OlK_JQFQPLFPP04e7R6EMBLR3q8o2E"
+ style='width:471.75pt;height:306pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/8/8f/UChicago2015Notebook_filesimage059.png" o:title="r6zsedRwioKWtxu0tkFeTi7D_42tqiJY0mG8k6qj8E7kMkYtRZW0w5TzpNGh4g2ICpc5CisNFak7tRlvsuaNWZSWdBa8hctZ45hCaNhC9OlK_JQFQPLFPP04e7R6EMBLR3q8o2E"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=629 height=408
+src="..//wiki/images/2/2b/UChicago2015Notebook_filesimage060.png"
+alt="https://lh5.googleusercontent.com/r6zsedRwioKWtxu0tkFeTi7D_42tqiJY0mG8k6qj8E7kMkYtRZW0w5TzpNGh4g2ICpc5CisNFak7tRlvsuaNWZSWdBa8hctZ45hCaNhC9OlK_JQFQPLFPP04e7R6EMBLR3q8o2E"
+v:shapes="Picture_x0020_30"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequencing Results</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequencing successful for MC004<span
+class=GramE>,5,7</span>. MC006 and MC007 samples probably <span class=SpellE>mislabelled</span>
+as sequencing indicates correct MC006 <span class=SpellE>phosphomimetic</span>
+is in MC007 sequence and vice versa. Otherwise, MC007<span class=GramE>,5</span>,
+and 4 are all correctly sequenced. Point mutation in MC006 (labelled MC007) samples
+base 244 of <span class=SpellE>Snapgene</span> file. Point mutation seems to be
+silent (GGC to GGT, translate features lists both as glycine. Four read out <span
+class=SpellE>KaiC</span> variants seem good to go. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC001 -still waiting for colony PCR/
+specific primers to arrive for assembly</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC002/3 -still waiting for TOPO kit to
+come for transformation ligation and assembly</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC004/7 -all assembled </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH001 -assembled, sequence verify </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH002 -<span class=SpellE>gblocks</span>
+not arrived yet</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH003 -assembled, sequence verify</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH004 -<span class=SpellE>gblocks</span>
+not arrived</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Zero-Blunt TOPT Ligation and Transformation</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>We will be ligating and transforming four samples in order to
+allow the bacteria to amplify blocks 1 of MC002 and MC003. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;mso-list:l8 level1 lfo68;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>MC002_b1<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l8 level1 lfo68;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>MC003_b1<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l8 level1 lfo68;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>MC002_b1 after
+     PCR<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l8 level1 lfo68;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>MC003_b1 after
+     PCR <o:p></o:p></span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ligation</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l20 level1 lfo69;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Combine 2uL product, 1 <span class=SpellE>uL</span> provided salt
+solution from <span class=SpellE>pTOPO</span> kit, 2uL H20, 1 <span
+class=SpellE>uL</span> <span class=SpellE>pCR</span> II-Blunt-TOPO vector <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l20 level1 lfo69;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Pipette up and down a few times to mix. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l20 level1 lfo69;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate 5 min at room temperature. &nbsp;&nbsp;<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l20 level1 lfo69;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>No overnight incubation needed! &nbsp;But you can leave it
+overnight at 4°C if you cannot proceed with the transformation right away, <o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Thaw one vial of Chemically Competent E. coli cells per
+transformation on ice. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 2&#956;l ligation product to the thawed cells. &nbsp;Keep on
+ice for 30 mins. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Transfer vials to a 42°C water bath for 45 seconds. Immediately
+return the tubes to ice for 2 min. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 250&#956;l room temperature SOC <span class=SpellE>orLB</span>
+to each vial. &nbsp;&nbsp;<o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate for 1 <span class=SpellE>hr</span> at 37°C. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>6.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Plate 200&#956;l cells on LB + kanamycin plates. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l4 level1 lfo70;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>7.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Incubate overnight at 37°C<o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>&nbsp;*The vector confers resistance to
+kanamycin, NOT ampicillin. &nbsp;</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span class=GramE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>pTOPO</span></span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> kit) </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>&nbsp;&nbsp;Note: if you do not have a
+sufficient number of <span class=SpellE>tranformants</span> in 200&#956;l,
+repeat the transformation, do a short spin (20</span></i><i><span
+style='font-size:16.0pt;font-family:"Cambria Math",serif;mso-bidi-font-family:
+"Cambria Math";color:black'>&#8208;</span></i><i><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>30 sec) to
+gently pellet your cells just before plating. &nbsp;Remove 100&#956;l of the
+supernatant, <span class=SpellE>resuspend</span> the cells by <span
+class=SpellE>pipeting</span> up and down.</span></i><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Set up PCR
+for amplifying MC002_b1 and MC003_b1. The products from this amplification
+(correct size ~1850-1970 bps) will be ligated and transformed using the TOPO
+zero blunt kit in order to have more specific inserts. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Set
+up 2 50uL 1X PCR Reactions:</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span class=SpellE><span
+     style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+     Arial'>dNTPS</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+     mso-bidi-font-family:Arial'>: 1uL<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>H2O: 31 <span
+     class=SpellE>uL</span><o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DNAP: 0.5 <span
+     class=SpellE>uL</span><o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DNA: 1 <span
+     class=SpellE>uL</span> (<span class=SpellE>gblocks</span> MC002_b1 and
+     MC003_b1)<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DMSO: 1.5uL <o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>HF Buffer: 10uL<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>F Primer: 2.5uL
+     (MC029)<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l7 level1 lfo71;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>R Primer: 2.5 <span
+     class=SpellE>uL</span> (MC010 for MC002_b1 and MC014 for MC003_b1)<o:p></o:p></span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings for <span
+class=SpellE>gBlock</span> PCR</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>66°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1.65 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Innoculation</span></b></span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> </span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Colonies from MC001 plates were inoculated as colony PCR results
+were ambiguous. Samples: 115,116,117,118,125,126,127,128. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l6 level1 lfo72;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Aliquot enough LB for 2mL/sample into a Falcon tube. Pipette 2mL
+of LB into each culture tube/sample. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l6 level1 lfo72;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Pipette all of the colony suspension into the culture tube as
+well. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l6 level1 lfo72;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Put in antibiotic to act as primary screening for insert -Cam 1uL,
+Amp 4uL (antibiotics stored in the -20 fridge) <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l6 level1 lfo72;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Place culture tubes into shaker at 37C overnight (~16 <span
+class=SpellE>hrs</span>) <o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>1% agarose gel used to run gel electrophoresis for MC002_b1 and
+MC003_b1. Gel run under 120V for 30 mins. Gel extraction of estimated product
+size conducted under blue light. 5uL 1kB Plus Invitrogen ladder loaded. 50uL of
+samples loaded. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_31"
+ o:spid="_x0000_i1035" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/gsdssnTT8wTdkdP-jkjDqH53bh0FIYeCHl3zd9dceMtuoFXS-9xw9sbQjkiysSL3jwAAWkBECqm1X73yezacNKHin-BvvY7_Skq4KIT-tLDWULzTq83bSFnzUBhfV1zma2itdqg"
+ style='width:351pt;height:456.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/0/04/UChicago2015Notebook_filesimage061.png" o:title="gsdssnTT8wTdkdP-jkjDqH53bh0FIYeCHl3zd9dceMtuoFXS-9xw9sbQjkiysSL3jwAAWkBECqm1X73yezacNKHin-BvvY7_Skq4KIT-tLDWULzTq83bSFnzUBhfV1zma2itdqg"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=468 height=609
+src="..//wiki/images/e/e1/UChicago2015Notebook_filesimage062.png"
+alt="https://lh6.googleusercontent.com/gsdssnTT8wTdkdP-jkjDqH53bh0FIYeCHl3zd9dceMtuoFXS-9xw9sbQjkiysSL3jwAAWkBECqm1X73yezacNKHin-BvvY7_Skq4KIT-tLDWULzTq83bSFnzUBhfV1zma2itdqg"
+v:shapes="Picture_x0020_31"><![endif]></span><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/19/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_32"
+ o:spid="_x0000_i1034" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/53EDmknNqpgNN_Znj7zqGc_ubMFs6HmH7Jaw3E17fp-VLhATtdq4jkvgPowy2xAbhDzIl4UmAtpkMNxej-RseZJQdifLyzPOInGIYLkWlSip8j6bfLlEJEkoFV-unPToWnq4i9w"
+ style='width:720.75pt;height:616.5pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/e/ea/UChicago2015Notebook_filesimage063.png" o:title="53EDmknNqpgNN_Znj7zqGc_ubMFs6HmH7Jaw3E17fp-VLhATtdq4jkvgPowy2xAbhDzIl4UmAtpkMNxej-RseZJQdifLyzPOInGIYLkWlSip8j6bfLlEJEkoFV-unPToWnq4i9w"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=961 height=822
+src="..//wiki/images/7/77/UChicago2015Notebook_filesimage064.png"
+alt="https://lh4.googleusercontent.com/53EDmknNqpgNN_Znj7zqGc_ubMFs6HmH7Jaw3E17fp-VLhATtdq4jkvgPowy2xAbhDzIl4UmAtpkMNxej-RseZJQdifLyzPOInGIYLkWlSip8j6bfLlEJEkoFV-unPToWnq4i9w"
+v:shapes="Picture_x0020_32"><![endif]></span><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformation Results</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Bacterial lawns seen on all plates. Perhaps issue with plates or
+competent cells are naturally resistant to kanamycin? Will try dilutions if
+cells are very efficient as cells could also be very conducive to
+transformation. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br>
+<br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequencing Results</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Analyzed samples 124 and 121 using Blast and chromatograms. Both
+samples did not have whole insert -MC031 gave no results for both samples. In
+both samples sequencing only at end of insert (part of <span class=SpellE>KaiC</span>
+w/<span class=SpellE>KaiB</span> and suffix) resulted.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Miniprep</span></b></span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>-</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+class=SpellE><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Miniprep</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> inoculations 115,116,117,118,125,126,127,128.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>1.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Centrifuge cells at 12000 <span class=SpellE>rcf</span> for 3 mins
+to harvest cells. Remove all medium. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>2.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+class=SpellE><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Resuspend</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> cells by adding 250uL of resuspension buffer R3 with RNase A. Mix
+up and down until homogenous. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>3.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 250uL of Lysis buffer (L7) to lyse cells. Mix gently by
+inverting capped <span class=SpellE>tube.Do</span> not vortex, incubate at room
+temp for 5 mins. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>4.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 350uL of precipitation buffer N4. <span class=GramE>mix</span>
+immediately by inverting tube or vigorously shaking. Do not vortex, centrifuge
+at 20,000 <span class=SpellE>rcfs</span> for 10 mins. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>5.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Load supernatant (750 <span class=SpellE>uL</span>) into Spin
+column (<span class=SpellE>machery</span> <span class=SpellE>nagel</span> used)
+and collection tube. Centrifuge column at 12,000 <span class=SpellE>rcfs</span>
+for 1 min. Discard flow through and place column back in wash tube. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>6.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 500 <span class=SpellE>uL</span> of wash buffer W10 with
+ethanol. Incubate at room temp for 1 min, centrifuge column at 12,000gs for 1
+min. Discard flow through. <i>(<b>*Optional Wash step</b></i><b>)</b><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>7.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Add 700 <span class=SpellE>uL</span> of wash buffer W9 with
+ethanol. <span class=GramE>centrifuge</span> column at 12,000 <span
+class=SpellE>gs</span> for 1 min. Discard flow through then dry spin for 1 min
+at 12,000 <span class=SpellE>gs</span>. Discard flow through. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>8.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Place spin column into microfuge tube. Add 50uL of TE Buffer to
+center of column. Incubate column in room temp for 1 min. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l18 level1 lfo73;
+tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+Garamond;mso-bidi-font-family:Garamond;color:black'><span style='mso-list:Ignore'>9.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Centrifuge column at 12,000 <span class=SpellE>rcfs</span> for 2
+mins. Discard column. <span class=SpellE>Nanodrop</span> DNA. Store at 4C short
+term, -20C long term. <o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> results-</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>115: 91.8 ng/<span class=SpellE>uL</span>,
+/280: 1.90</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>116: 172.0 ng/<span class=SpellE>uL</span>,
+/280: 1.82</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>117: 152.0 ng/<span class=SpellE>uL</span>,
+/280: 1.91</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>118: 120.4 ng/<span class=SpellE>uL</span>,
+/280: 1.81</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>125: 187.0 ng/<span class=SpellE>uL</span>,
+/280: 1.88</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>126: 149.2 ng/<span class=SpellE>uL</span>,
+/280: 1.88</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>127: 211.0 ng/<span class=SpellE>uL</span>,
+/280: 1.87</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>128: 222.8 ng/<span class=SpellE>uL</span>,
+/280: 1.89</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Set up PCR for both <span class=SpellE>miniprep</span> PCR and PCR
+of MC002_b1 and MC003_b1 to redo ligation and transformation. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up 2 50uL 1X PCR Reactions for
+MC002_b1 and MC003_b1:</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span class=SpellE><span
+     style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+     Arial'>dNTPS</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+     mso-bidi-font-family:Arial'>: 1uL<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>H2O: 31 <span
+     class=SpellE>uL</span><o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DNAP: 0.5 <span
+     class=SpellE>uL</span><o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DNA: 1 <span
+     class=SpellE>uL</span> (<span class=SpellE>gblocks</span> MC002_b1 and
+     MC003_b1)<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>DMSO: 1.5uL <o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>HF Buffer: 10uL<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>F Primer: 2.5uL
+     (MC029)<o:p></o:p></span></li>
+ <li class=MsoNormal style='color:black;mso-list:l12 level1 lfo74;tab-stops:
+     list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
+     font-family:"Garamond",serif;mso-bidi-font-family:Arial'>R Primer: 2.5 <span
+     class=SpellE>uL</span> (MC010 for MC002_b1 and MC014 for MC003_b1)<o:p></o:p></span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Made 9/5x of 1X PCR Master Mix for 10uL
+samples (used 9.8 <span class=SpellE>uL</span> Mix and 0.2 <span class=SpellE>uL</span>
+DNA):</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>HF
+Buffer: 18.00</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DMSO:
+.70 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span><span
+class=SpellE><span class=GramE>dNTPS</span></span>: 1.80</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>H2O:
+.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DNAP:
+.25 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Used
+.50 <span class=SpellE>uL</span> of 10X MC003 and MC004 for mix. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings (used 2
+thermocyclers, one for 10uL samples and other for 50uL samples)-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>67°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1:40 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>1% agarose
+gel run at 120V for 30 mins. Loaded 10uL and 45 <span class=SpellE>uL</span>
+for <span class=SpellE>minipreps</span> and MC002/3 gel extractions
+respectively. Loaded 5uL of 1kB Plus Invitrogen ladder. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image- </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="Picture_x0020_33" o:spid="_x0000_i1033" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/RA4hfJiLGXdMws--G_SPC1NtjjUnOEMIMePRoQnqebfDInZavx4jESYy7N2sKuI9OkX5pfQmUqO3CwG6U8Ov2Okpu8fqSNkS8_uUcBuXezahf8jMUUTHxpdV_i_ekVmKz7O-7_s"
+ style='width:885pt;height:628.5pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/7/7f/UChicago2015Notebook_filesimage065.png" o:title="RA4hfJiLGXdMws--G_SPC1NtjjUnOEMIMePRoQnqebfDInZavx4jESYy7N2sKuI9OkX5pfQmUqO3CwG6U8Ov2Okpu8fqSNkS8_uUcBuXezahf8jMUUTHxpdV_i_ekVmKz7O-7_s"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1180 height=838
+src="..//wiki/images/5/59/UChicago2015Notebook_filesimage066.png"
+alt="https://lh6.googleusercontent.com/RA4hfJiLGXdMws--G_SPC1NtjjUnOEMIMePRoQnqebfDInZavx4jESYy7N2sKuI9OkX5pfQmUqO3CwG6U8Ov2Okpu8fqSNkS8_uUcBuXezahf8jMUUTHxpdV_i_ekVmKz7O-7_s"
+v:shapes="Picture_x0020_33"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/20</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequences Submitted</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC001 samples submitted in morning for
+sequencing. 4uM primer stock made by diluting in water (1:24 <span
+class=SpellE>primer<span class=GramE>:water</span></span> ratio). 10 <span
+class=SpellE>uL</span> of <span class=SpellE>miniprepped</span> DNA submitted
+with 10uL of 4uM primers. Samples 115,117, 125, 127, 128 sent. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Primers used: </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>VF2 (1)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC003 (2)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC031 (3)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC032 (4)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC006 (5)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC033 (6)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC008 (7)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>VR (8) &nbsp;</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson Assembly</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>New primers for MC001 arrived.
+Proceeded with Gibson <span class=SpellE>assesmbly</span>. Incubated mix at 50C
+for 1 hour.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_34" o:spid="_x0000_i1032"
+ type="#_x0000_t75" alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"
+ style='width:1020.75pt;height:60.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage067.png" o:title="nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1361 height=81
+src="..//wiki/images/0/03/UChicago2015Notebook_filesimage068.png"
+alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"
+v:shapes="Picture_x0020_34"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up PCR for MC002_b1, MC003_b1,
+MC001_b1, MC001_b2, and MC001 Gibson products. Total 7 reaction (MC002/3 block
+s sampled twice as one will be used for gel extraction then TOPO
+ligation/transformation, the other will be used to amplify a second time). </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span class=SpellE><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Phusion</span></span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'> 7.5 x of 1X mix</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>HF Buffer -75.00 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DMSO -11.25 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>DNTPs -7.50 <span class=SpellE>uL</span>
+</span><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span>H2O -232.50 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'><span
+style='mso-tab-count:1'>          </span><span class=SpellE>Phusion</span> DNAP
+-3.75 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 44uL of PCR Mix to 2.5 of F and R
+Primer each and 1 <span class=SpellE>uL</span> of DNA</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA :</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> Primers</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC002_<span class=GramE>b1 :</span>
+MC029, MC010</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC003_<span class=GramE>b1 :</span>
+MC029, MC014</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson <span class=SpellE><span
+class=GramE>rxn</span></span><span class=GramE> :</span> 1b1F, 1b2R</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC001_<span class=GramE>b1 :</span>
+b1F, 1b1R</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC002_b2: &nbsp;1b2F, 1b2R </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings:</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings (used 2
+thermocyclers, one for 10uL samples and other for 50uL samples)-</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>66°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1:30 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel <span class=SpellE>Electrohporesis</span>-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>1% Agarose gel run at 120V for 30 mins. 45 <span class=SpellE>uL</span>
+of samples loaded. Hard to see bands under blue light. Used UV light for quick
+clarification of size. No products from MC001 Gibson seen. MC001_b1 and
+MC001_b2 extracted separately. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/21</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Due to failure of yesterday’s PCR, gradient PCR was set up in
+order to determine optimal anneal temperature. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Master <span class=GramE>mix</span> of
+<span class=SpellE>uL</span> created (calculate ratio of 1X x 7.5/5)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 15 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 1.5 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 0.75 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O = 46.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 2.25 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA =1.5 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC003= 3.75 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC004 =3.75 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>10uL Master mix aliquoted into 7
+samples.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98<sup>o</sup> 2
+min, 98<sup>o</sup> 15 s, 60<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>,
+<sup>o</sup>, 68<sup>o</sup>, 70<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup>
+min, 72<sup>o</sup> 10 min, 4<sup>o</sup> hold.</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>,
+.0<sup>o</sup>, 63.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>,
+.0<sup>o</sup></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup>
+for 30 cycles</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Purification </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>MC001_b1 and MC001_b2 extracted yesterday were purified. Used <span
+class=SpellE>Machery</span>-Nagel protocol. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Weights-</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1b1 -13.4 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1b2 - 133.4 mg</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> results-</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1b1 - 13.1 ng/<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1b2 -19.2 ng/<span class=SpellE>ul</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>1% agarose gel run for gradient PCR. 120V for 30 mins. 1kB
+Invitrogen ladder used. 10uL of sample loaded. Expected product size ~3000 bps.
+No expected product size appeared on gel. Perhaps indicates issues with Gibson
+assembly. &nbsp;24 inoculated colony PCRs also run on separate gel. 3 colonies
+chosen for inoculation and <span class=SpellE>miniprep</span>: 3<span
+class=GramE>,9</span> and 22. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_35"
+ o:spid="_x0000_i1031" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/E5f_qehJxo9MhfwKRgs8YHVlaEh966MaCTP4Xq-KzMGrBoRrVE-LcC-a1YttvHeNVq-9wOmklO8kMx6046adLR488kWfBiTcmmOnmTdqtlgv-nQ4_ALIa1xcrAR1lD9RVmpuWm0"
+ style='width:592.5pt;height:377.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/6/6b/UChicago2015Notebook_filesimage069.png" o:title="E5f_qehJxo9MhfwKRgs8YHVlaEh966MaCTP4Xq-KzMGrBoRrVE-LcC-a1YttvHeNVq-9wOmklO8kMx6046adLR488kWfBiTcmmOnmTdqtlgv-nQ4_ALIa1xcrAR1lD9RVmpuWm0"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=790 height=503
+src="..//wiki/images/f/f2/UChicago2015Notebook_filesimage070.png"
+alt="https://lh5.googleusercontent.com/E5f_qehJxo9MhfwKRgs8YHVlaEh966MaCTP4Xq-KzMGrBoRrVE-LcC-a1YttvHeNVq-9wOmklO8kMx6046adLR488kWfBiTcmmOnmTdqtlgv-nQ4_ALIa1xcrAR1lD9RVmpuWm0"
+v:shapes="Picture_x0020_35"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_36"
+ o:spid="_x0000_i1030" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/sUvtKnAEk2sK3xSabkXHLEKeeMi2FUX_zR6SwqW8TfR27XVWG-gk72KBOTbFM87FEBjAzef8k1hNyPRYitVYszT-EDvgJhlVi4C2qAWWzk4WdmgZahr0sqhXlqm0Z-YQnCo6gfo"
+ style='width:381.75pt;height:497.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/7/78/UChicago2015Notebook_filesimage071.png" o:title="sUvtKnAEk2sK3xSabkXHLEKeeMi2FUX_zR6SwqW8TfR27XVWG-gk72KBOTbFM87FEBjAzef8k1hNyPRYitVYszT-EDvgJhlVi4C2qAWWzk4WdmgZahr0sqhXlqm0Z-YQnCo6gfo"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=509 height=663
+src="..//wiki/images/5/5b/UChicago2015Notebook_filesimage072.png"
+alt="https://lh4.googleusercontent.com/sUvtKnAEk2sK3xSabkXHLEKeeMi2FUX_zR6SwqW8TfR27XVWG-gk72KBOTbFM87FEBjAzef8k1hNyPRYitVYszT-EDvgJhlVi4C2qAWWzk4WdmgZahr0sqhXlqm0Z-YQnCo6gfo"
+v:shapes="Picture_x0020_36"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gibson- </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>Gibson of MC001 repeated. Incubated mix at 50C for 1 hour. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><br>
+<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_37"
+ o:spid="_x0000_i1029" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"
+ style='width:1020.75pt;height:60.75pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage067.png" o:title="nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1361 height=81
+src="..//wiki/images/0/03/UChicago2015Notebook_filesimage068.png"
+alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"
+v:shapes="Picture_x0020_37"><![endif]></span><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR- </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>PCR set up for MC001 blocks and MC001 8/21 Gibson. 6 samples in
+total. 1X PCR Mix for each sample set up:</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DMAP:
+.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DMSO:
+.5 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span><span
+class=SpellE><span class=GramE>dNTPS</span></span>: 1 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>DNA:
+uL</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>HF
+Buffer: 10 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>H2O:
+<span class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>F
+Primer: 2.5 <span class=SpellE>uL</span> (10uM)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>R
+Primer: 2.5 <span class=SpellE>uL</span> (10uM) </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>DNA (Primers): MC001_b1
+(MC001_b1_F_new, MC001_b1_R_new), MC001_b1 (MC001_b2_F_new, MC001_b2_R_new),
+MC001 Gibson (MC001_b1_F_new, MC001_b2_R_new)</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR Conducted one at high anneal
+temperature (68C) one at low anneal temperature (64C)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler Settings (used 2 thermocyclers,
+one for 10uL samples and other for 50uL samples)-</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-right-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>STEP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:.75pt;
+  mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TEMP </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;mso-border-left-alt:
+  solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-left-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><b><u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  Arial;color:#4C4C4C;background:white'>TIME </span></u></b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Initial Denaturation</span></u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>98°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>30 seconds </span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>30 Cycles</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>98°C</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>68 or 64°C </span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>10 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  mso-bidi-font-family:Arial;color:#4C4C4C;background:white'>30 seconds</span></u><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+  "Times New Roman"'><o:p></o:p></span></p>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>1:30 mins</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:3'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>Final Extension</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>72°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:#ECEDE8'>10 min</span></u><span style='font-size:16.0pt;
+  font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:4;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;mso-border-top-alt:
+  solid #B3B3B3 .25pt;mso-border-alt:solid #B3B3B3 .25pt;mso-border-right-alt:
+  solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-top-alt:.25pt;mso-border-left-alt:
+  .75pt;mso-border-bottom-alt:.25pt;mso-border-right-alt:.75pt;mso-border-color-alt:
+  #B3B3B3;mso-border-style-alt:solid;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>4°C</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;mso-border-top-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;mso-border-alt:solid #B3B3B3 .25pt;
+  mso-border-left-alt:solid #B3B3B3 .75pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal style='mso-line-height-alt:0pt'><u><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#4C4C4C;
+  background:white'>Hold</span></u><span style='font-size:16.0pt;font-family:
+  "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span></span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>1% agarose gel run at 120V for 30 mins. 1kB Plus <span
+class=SpellE>invitrogen</span> ladder used. 50uL of samples loaded. Results
+very inconclusive. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image-</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/24</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Miniprep</span></b></span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'>-</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'><span style='mso-tab-count:1'>          </span>Inoculated
+TOPO MC002_b1 and MC003_b1 samples <span class=SpellE>miniprepped</span> using <span
+class=SpellE>invitrogen</span> protocol. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Miniprep</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> inoculations 115,116,117,118,125,126,127,128.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo75;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Centrifuge cells at 12000 <span
+class=SpellE>rcf</span> for 3 mins to harvest cells. Remove all medium. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo76;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span class=SpellE><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Resuspend</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> cells by adding 250uL of resuspension buffer R3 with RNase A. Mix
+up and down until homogenous. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo77;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 250uL of Lysis buffer (L7) to lyse
+cells. Mix gently by inverting capped <span class=SpellE>tube.Do</span> not
+vortex, incubate at room temp for 5 mins. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo78;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 350uL of precipitation buffer N4. <span
+class=GramE>mix</span> immediately by inverting tube or vigorously shaking. Do
+not vortex, centrifuge at 20,000 <span class=SpellE>rcfs</span> for 10 mins. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo79;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Load supernatant (750 <span
+class=SpellE>uL</span>) into Spin column (<span class=SpellE>machery</span> <span
+class=SpellE>nagel</span> used) and collection tube. Centrifuge column at
+,000 <span class=SpellE>rcfs</span> for 1 min. Discard flow through and place
+column back in wash tube. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo80;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 500 <span class=SpellE>uL</span> of
+wash buffer W10 with ethanol. Incubate at room temp for 1 min, centrifuge
+column at 12,000gs for 1 min. Discard flow through. <i>(<b>*Optional Wash step</b></i><b>)</b><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo81;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Add 700 <span class=SpellE>uL</span> of
+wash buffer W9 with ethanol. <span class=GramE>centrifuge</span> column at
+,000 <span class=SpellE>gs</span> for 1 min. Discard flow through then dry
+spin for 1 min at 12,000 <span class=SpellE>gs</span>. Discard flow through. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo82;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Place spin column into microfuge tube.
+Add 50uL of TE Buffer to center of column. Incubate column in room temp for 1
+min. <o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;mso-list:l1 level1 lfo83;
+vertical-align:baseline'><![if !supportLists]><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:Garamond;mso-bidi-font-family:
+Garamond;color:black'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span></span><![endif]><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Centrifuge column at 12,000 <span
+class=SpellE>rcfs</span> for 2 mins. Discard column. <span class=SpellE>Nanodrop</span>
+DNA. Store at 4C short term, -20C long term. <o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Nanodrop</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> results-</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A21: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A22: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A31:</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>A32: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>B21: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>B22: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>B31: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>B32: </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>None were sufficient enough to send for
+sequencing. Likely errors also due to overgrowth of colonies. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br>
+<br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>PCR </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gradient PCR of only MC001 blocks
+conducted. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Master <span class=GramE>mix</span> of
+<span class=SpellE>uL</span> created (calculate ratio of 1X x 7.5/5)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> 5X HF Buffer = 37.5 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>dNTPs 10 <span
+class=SpellE>mM</span> &nbsp;= 3.75 &nbsp;<span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span class=SpellE><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> DNAP = 1.875 <span class=SpellE>uL</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>H<sub>2</sub>O =
+.25 <span class=SpellE>uL</span></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DMSO = 5.625 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>DNA =3.75 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>F Primer MC003= 9.375 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=GramE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Courier New";color:black'>o</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman";
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>R Primer MC004 =9.375 <span
+class=SpellE>uL</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>10uL Master mix aliquoted into 7
+samples.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Thermocycler settings- 98<sup>o</sup> 2
+min, 98<sup>o</sup> 15 s, 60<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>,
+<sup>o</sup>, 68<sup>o</sup>, 70<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup>
+min, 72<sup>o</sup> 10 min, 4<sup>o</sup> hold.</span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>,
+.0<sup>o</sup>, 63.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>,
+.0<sup>o</sup></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup>
+for 30 cycles</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Gel Electrophoresis</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>1 % agarose gel run for 30 mins at
+V. 25 <span class=SpellE>uL</span> of samples loaded. 1kB plus Invitrogen
+ladder loaded. Only sufficient product seen for block 2. Indicates issues with
+block 1. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_38"
+ o:spid="_x0000_i1028" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/vNACBLm6LidZIh48dOsbDGB6YusPmNCCVzU9Ima_K2Ma8PKbDmeuKbwyH2ZEr69jrCBsMfySsW7latn7jNYFWg66N1xdoZvNoYlJz9EYupv2N3ohTwqP2HvJZ-6Di04qAEWVe_0"
+ style='width:461.25pt;height:335.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/6/6e/UChicago2015Notebook_filesimage073.png" o:title="vNACBLm6LidZIh48dOsbDGB6YusPmNCCVzU9Ima_K2Ma8PKbDmeuKbwyH2ZEr69jrCBsMfySsW7latn7jNYFWg66N1xdoZvNoYlJz9EYupv2N3ohTwqP2HvJZ-6Di04qAEWVe_0"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=615 height=447
+src="..//wiki/images/7/79/UChicago2015Notebook_filesimage074.png"
+alt="https://lh3.googleusercontent.com/vNACBLm6LidZIh48dOsbDGB6YusPmNCCVzU9Ima_K2Ma8PKbDmeuKbwyH2ZEr69jrCBsMfySsW7latn7jNYFWg66N1xdoZvNoYlJz9EYupv2N3ohTwqP2HvJZ-6Di04qAEWVe_0"
+v:shapes="Picture_x0020_38"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/25</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Sequencing Results: Found a sample
+(127) containing entire MC001 insert! </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Innoculated</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> sample 127 and prepared for induction with L-<span class=SpellE>Rhamnose</span>.
+</span><span style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:
+"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Re-plated TOPO reactions for MC002/3 as
+overgrowth of colonies on previous plates. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up inductions however, cultured at
+C instead of 30C. Pay attention to this, this is important, all the protocols
+have induced L-<span class=SpellE>Rhamnose</span> at 30C </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/26</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Start over the inductions, follow the <span
+class=SpellE>igem</span> protocol file for help. You will need to make more LB.
+I have already made a few glycerol stocks. You will need to set up a 2mL <span
+class=SpellE>innoculation</span> in a culture tube as well as a 40mL <span
+class=SpellE>innoculation</span> in a flask. The 40mL <span class=SpellE>innoculation</span>
+will be used for inductions. [Finished]</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Redo TOPO transformations. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/27</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/28</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>8/31</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Reviewed work done over past three
+days. Discussed ways to resolve western blot issue. Seems to be a lot of
+non-specific binding -we see blots of many different sizes. Blots of <span
+class=SpellE>antibodied</span> products are very bright relative to ladder.
+Kevin mentioned degradation could be an issue (would cause lots of different
+sizes in blots). On closer examination, 0% <span class=SpellE>rhamnose</span>
+has no (barely any) protein. Updated notebook, e-mailed Justin and Kevin for
+trouble shooting. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH- Type up lysing assay and <span
+class=SpellE><span class=GramE>bradford</span></span> assay -give some
+specifics about western blot (how much each sample, and calculations) </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>E-mail sleep people</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/1</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Induction Solutions</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>In order to re-do induction western
+blots, made new 10% <span class=SpellE>Rhamnose</span> w/v solution by diluting
+g of <span class=SpellE>Rhamnose</span> in 9 mL of water to bring final
+volume to 10mL.</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Culture</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up two 40 mL cultures of sample 127
+in 250 mL flasks. Added 20uL of Cam in each, 40 mL of LB and 10uL of 2 mL
+sample 127 overnight culture. Note: 2mL overnight culture has been stored in
+room temperature for past four days -may also have to start from new glycerol
+stock. 40mL cultures placed in 37C incubator at 11:50. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/2</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Dilutions of <span class=SpellE>Rhamnose</span>
+Inducer Samples</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>As discussed in meetings, errors with
+previous western blot likely to be caused due to overexpression of <span
+class=SpellE>pRhamnose</span>. <span class=SpellE><span class=GramE>iGEM</span></span>
+team used low copy number plasmid. Re-made inducer solutions using 1/10 of %
+from 0.001 - 0.1% in order to test lower gradient. Induction started at 2:30 am
+when OD600 of 40 mL <span class=SpellE>innoculations</span> was 1.6(?) </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Minimal Media</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ordered M9 minimal media 5X salts on
+Rust Lab tab. <span class=GramE>Should</span> take till <span class=SpellE>friday</span>
+to arrive. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Restriction Digest </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Protocol from <span class=SpellE>iGEM</span>.</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:10.0pt;mso-outline-level:2'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+"Times New Roman";mso-bidi-font-family:Arial;color:#282828;background:white'>Digest</span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></b></p>
+<p class=MsoNormal style='margin-top:3.0pt;margin-right:0in;margin-bottom:0in;
+margin-left:53.25pt;margin-bottom:.0001pt;text-indent:-.25in;mso-list:l17 level1 lfo84;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Enzyme Master Mix for Plasmid Backbone (25ul
+total, for 5 <span class=SpellE>rxns</span>)</span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>5 <span class=SpellE>ul</span> NEB Buffer 2
+(use CutSmart)</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>0.5 <span class=SpellE>ul</span> BSA</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>0.5 <span class=SpellE>ul</span> </span><a
+href="http://www.neb.com/nebecomm/products/productR3101.asp"><span
+class=SpellE><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#002BB8;background:white'>EcoRI</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#002BB8;background:white'>-HF</span></a><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>0.5 <span class=SpellE>ul</span> </span><a
+href="http://www.neb.com/nebecomm/products/productR0140.asp"><span
+class=SpellE><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#002BB8;background:white'>PstI</span></span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>0.5 <span class=SpellE>ul</span> </span><a
+href="http://www.neb.com/nebecomm/products/productR0176.asp"><span
+class=SpellE><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#002BB8;background:white'>DpnI</span></span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'> (Used to digest any template DNA from
+production)</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l17 level2 lfo85;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>18 <span class=SpellE>ul</span> dH20</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;mso-list:l13 level1 lfo86;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Digest Plasmid Backbone</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l13 level2 lfo87;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add 4 <span class=SpellE>ul</span> linearized
+plasmid backbone (25ng/<span class=SpellE>ul</span> for 100ng total)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;mso-list:
+l13 level2 lfo87;tab-stops:list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add 4 <span class=SpellE>ul</span> of Enzyme
+Master Mix</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:2.0pt;
+margin-left:104.25pt;text-indent:-.25in;mso-list:l13 level2 lfo87;tab-stops:
+list 1.0in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Digest 37C/30 min, heat kill 80C/20 min</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:10.0pt;mso-outline-level:2'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+"Times New Roman";mso-bidi-font-family:Arial;color:#282828;background:white'>Ligation</span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-fareast-font-family:
+"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></b></p>
+<p class=MsoNormal style='margin-top:3.0pt;margin-right:0in;margin-bottom:0in;
+margin-left:53.25pt;margin-bottom:.0001pt;text-indent:-.25in;mso-list:l10 level1 lfo88;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add 2ul of digested plasmid backbone (25 ng)</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;mso-list:l10 level1 lfo88;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add 1 <span class=SpellE>ul</span> </span><a
+href="http://www.neb.com/nebecomm/products/productm0202.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#002BB8;background:white'>T4 DNA ligase buffer</span></a><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>. <b>Note:</b> Do not use quick ligase</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;mso-list:l10 level1 lfo88;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add 0.5 <span class=SpellE>ul</span> </span><a
+href="http://www.neb.com/nebecomm/products/productm0202.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#002BB8;background:white'>T4 DNA ligase</span></a><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;mso-list:l10 level1 lfo88;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Add water to 10 <span class=SpellE>ul</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;mso-list:l10 level1 lfo88;
+tab-stops:list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Ligate 16C/30 min, heat kill 80C/20 min</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:1.0pt;
+margin-left:53.25pt;text-indent:-.25in;mso-list:l10 level1 lfo88;tab-stops:
+list .5in;background:white;vertical-align:baseline'><![if !supportLists]><span
+style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Wingdings;
+mso-fareast-font-family:Wingdings;mso-bidi-font-family:Wingdings;color:#282828'><span
+style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:#282828;background:white'>Transform with 1-2 <span class=SpellE>ul</span>
+of product</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:#282828'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Restreaked</span></b></span><b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> TOPO plate</span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Plates of successful TOPO reactions for
+MC002_b1 and MC003_b1. The colonies on these plates had overgrown -so in an
+attempt to isolate one colony for <span class=SpellE>miniprepping</span>, these
+colonies were streaked onto new Cam plates. &nbsp;&nbsp;</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transformed new TOPO reactions for
+MC02/3 and BH002/3</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>New TOPO reactions were conducted in
+parallel with the streaking. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up Gibson of BH002/3</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>BH002 and BH003 were <span
+class=SpellE>gibsonsed</span> with their respective Amp backbones and <span
+class=SpellE>Cam</span> backbones. </span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/3 </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Lysed cells after induction </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Cells were lysed after 10 hours of
+induction. Cells <span class=SpellE>resuspened</span> in lysis buffer,
+transferred to microfuge tube with 0.1 mL glass beads, vortex for 30 s and <span
+class=SpellE>incuabted</span> on ice for 1 min (repeated 5 times). </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford Assay </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford assay conducted to <span
+class=SpellE>quanity</span> amount of protein added. 1 mL 1x <span
+class=SpellE>bio-rad</span> protein reagent and 0.2 <span class=SpellE>uL</span>
+of protein sample used for each spectrophotometer (595 nm) reading. BSA protein
+standards with fixed concentrations made and absorbance measured for generating
+standard curve (concentration vs absorbance). </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Conducted Western blot </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel set up with purified
+proteins, cyanobacterial lysate and <span class=SpellE>ecoli</span> vector only
+lysate (not used in this specific assay). Proteins purified and loaded on
+-20% gel in 2 fold <span class=SpellE>diluations</span> from 100 ng to 1.56
+ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins.
+Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer constructed with membrane
+sandwich -plastic latched case, two <span class=SpellE>whatman</span> papers,
+two sponges, membrane, <span class=GramE>gel</span>. Transfer apparatus run for
+.5 hours at 90 V. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Blocking and Staining</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Memabrane</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> blocked with 2% dry milk and TBST for 1 hour. Membrane pieces
+stained with primary Kai rabbit antibodies for 1.5 hours. Membrane washed with
+washing buffer for 10 mins for 3 times (30 mins total). Membrane stained with
+secondary goat antibody for 1 hour. Membrane <span class=SpellE>peices</span>
+washed with washing buffer for 10 mins for 3 times (30 mins total). Membrane
+imaged. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ran colony PCR of TOPO reactions</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Only MC003 TOPO worked. BH002/3 <span
+class=SpellE>Gibsons</span> worked, no TOPO <span class=GramE>results &nbsp;for</span>
+MC002, BH002, BH003. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Inoculated negative control</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Ecoli</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> with vector (Cam backbone) <span class=SpellE>innoculated</span>
+at 37C. 2 mL <span class=SpellE>innoculation</span> generated. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Image</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_39"
+ o:spid="_x0000_i1027" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/qi6z0XeDqFcoAaomNv3uJYjNWeOkAjg9dWljPlg474qH2PgOCbVWStI-wwSYG4AxtZKZRKqFVHE-YPjUVuc6RKDlC6wNrP-9EizaWbl7cTfvD8M3k3ggW97-kzMdkMMMVg=s1600"
+ style='width:687.75pt;height:209.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage075.png" o:title="qi6z0XeDqFcoAaomNv3uJYjNWeOkAjg9dWljPlg474qH2PgOCbVWStI-wwSYG4AxtZKZRKqFVHE-YPjUVuc6RKDlC6wNrP-9EizaWbl7cTfvD8M3k3ggW97-kzMdkMMMVg=s1600"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=917 height=279
+src="..//wiki/images/9/94/UChicago2015Notebook_filesimage076.png"
+alt="https://lh4.googleusercontent.com/qi6z0XeDqFcoAaomNv3uJYjNWeOkAjg9dWljPlg474qH2PgOCbVWStI-wwSYG4AxtZKZRKqFVHE-YPjUVuc6RKDlC6wNrP-9EizaWbl7cTfvD8M3k3ggW97-kzMdkMMMVg=s1600"
+v:shapes="Picture_x0020_39"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>KaiB</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> and <span class=SpellE>KaiA</span> gave no clear results. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/4/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Set up <span class=SpellE>Rhamnose</span>
+time course experiment and <span class=SpellE>rhamnose</span> gradient </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Rhamnose</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> time course experiment conducted in both M9 supplemented media
+(4% glycerol, 0.1% <span class=SpellE>casamino</span> acids, antibiotic) and
+LB. 40 mL culture grown overnight. Cells pelleted, washed with 1 mL water,
+pelleted then reintroduced into <span class=SpellE>Lb</span> or M9 media. Time
+course consisted of induction at 30C for 10 hours. 0.5% <span class=SpellE>Rhamnose</span>
+used. Time course experiment conducted in order to examine behavior of <span
+class=SpellE>KaiA</span> throughout induction. 2 mL of cells frozen every 2
+hours for 16 hours. Shocks also conducted for 1 hour and 6 hours. Shocks
+conducted to emulate synchronization method -incubation in M9 media with no
+supplements to limit ATP available to cells. Samples after shock collected at 1
+hour, 6 hours, and 62 hours. </span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Rhamnose</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> gradient assay also conducted, using smaller gradient with 10
+fold dilution. Induction conducted for 10 hours. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/7/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>MC003, BH002, and BH003 colonies were
+submitted for sequencing in <span class=SpellE>UChicago</span> sequencing
+facility with respective primers. MC003 used specific primers, BH002/3 used M13
+and M14 primers as they were in TOPO vector. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/8/15</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford Assay </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford assay conducted to <span
+class=SpellE>quanity</span> amount of protein added. 1 mL 1x <span
+class=SpellE>bio-rad</span> protein reagent and 0.2 <span class=SpellE>uL</span>
+of protein sample used for each spectrophotometer (595 nm) reading. BSA protein
+standards with fixed concentrations made and absorbance measured for generating
+standard curve (concentration vs absorbance). </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Conducted Western blot </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel set up with purified
+proteins, cyanobacterial lysate and <span class=SpellE>ecoli</span> vector only
+lysate. Proteins purified and loaded on 14-20% gel in 2 fold <span
+class=SpellE>diluations</span> from 100 ng to 1.56 ng. Proteins loaded with 3 x
+loading dye and denatured at 70C for 10 mins. Empty lanes loaded with 1 x
+loading dye. Gel run at 300 v for 22 mins. Kaleidoscope ladder used. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer constructed with membrane
+sandwich -plastic latched case, two <span class=SpellE>whatman</span> papers,
+two sponges, membrane, <span class=GramE>gel</span>. Transfer apparatus run for
+.5 hours at 90 V. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel set up with purified
+proteins, cyanobacterial lysate and <span class=SpellE>ecoli</span> vector only
+lysate (not used in this specific assay). Proteins purified and loaded on
+-20% gel in 2 fold <span class=SpellE>diluations</span> from 100 ng to 1.56
+ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins.
+Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/9</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Blocking and Staining</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Membrane blocked with 2% dry milk and
+TBST for 1 hour. Membrane pieces stained with primary Kai rabbit antibodies for
+.5 hours. Membrane washed with washing buffer for 10 mins for 3 times (30 mins
+total). Membrane stained with secondary goat antibody for 1 hour. Membrane
+pieces washed with washing buffer for 10 mins for 3 times (30 mins total).
+Membrane imaged. Blot only conducted for <span class=SpellE>KaiA</span> and <span
+class=SpellE>KaiC</span> as issues with obtaining <span class=SpellE>KaiB</span>
+antibodies. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="Picture_x0020_40" o:spid="_x0000_i1026" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/SPybomQoqbf7c2PeVmAFpbrVANHCm5VAEpe6J2nuDqeXm2T5Kqb7YxjUIcdDBmv3O0XWM3OPUHn9kOsBKej490C6Qzh3JiNuHbM_PP_34-EwtQ-q7bbUw4-4wBS-q__gxw=s1600"
+ style='width:1129.5pt;height:682.5pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/b/b0/UChicago2015Notebook_filesimage077.png" o:title="SPybomQoqbf7c2PeVmAFpbrVANHCm5VAEpe6J2nuDqeXm2T5Kqb7YxjUIcdDBmv3O0XWM3OPUHn9kOsBKej490C6Qzh3JiNuHbM_PP_34-EwtQ-q7bbUw4-4wBS-q__gxw=s1600"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1506 height=910
+src="..//wiki/images/3/30/UChicago2015Notebook_filesimage078.png"
+alt="https://lh6.googleusercontent.com/SPybomQoqbf7c2PeVmAFpbrVANHCm5VAEpe6J2nuDqeXm2T5Kqb7YxjUIcdDBmv3O0XWM3OPUHn9kOsBKej490C6Qzh3JiNuHbM_PP_34-EwtQ-q7bbUw4-4wBS-q__gxw=s1600"
+v:shapes="Picture_x0020_40"><![endif]></span><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/10</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Induction of <span class=SpellE>Rhamnose</span>
+with lower gradient started again. Finished western blot for Kai B from
+previous assay. Ran PCR of block MC002_b1, MC003_b1. Gel extracted blocks. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_41"
+ o:spid="_x0000_i1025" type="#_x0000_t75" alt="https://lh4.googleusercontent.com/xsJGDEr3-BGG075g4bYfif1LUKzwP9hUAn2Sg0E1ZY5Br9Gg5ypX0FwcqgmTkhKsaLjU-x3qLo1cQ7DPU7eP3PM3S-Eer2pKTI-AKrcDNy-yOVWIWyz8hqLYqSf0ovDkrg=s1600"
+ style='width:804.75pt;height:203.25pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="..//wiki/images/4/4d/UChicago2015Notebook_filesimage079.png" o:title="xsJGDEr3-BGG075g4bYfif1LUKzwP9hUAn2Sg0E1ZY5Br9Gg5ypX0FwcqgmTkhKsaLjU-x3qLo1cQ7DPU7eP3PM3S-Eer2pKTI-AKrcDNy-yOVWIWyz8hqLYqSf0ovDkrg=s1600"/>
+</v:shape><![endif]--><![if !vml]><img border=0 width=1073 height=271
+src="..//wiki/images/a/ae/UChicago2015Notebook_filesimage080.png"
+alt="https://lh4.googleusercontent.com/xsJGDEr3-BGG075g4bYfif1LUKzwP9hUAn2Sg0E1ZY5Br9Gg5ypX0FwcqgmTkhKsaLjU-x3qLo1cQ7DPU7eP3PM3S-Eer2pKTI-AKrcDNy-yOVWIWyz8hqLYqSf0ovDkrg=s1600"
+v:shapes="Picture_x0020_41"><![endif]></span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/11 </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford Assay </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Bradford assay conducted to <span
+class=SpellE>quanity</span> amount of protein added. 1 mL 1x <span
+class=SpellE>bio-rad</span> protein reagent and 0.2 <span class=SpellE>uL</span>
+of protein sample used for each spectrophotometer (595 nm) reading. BSA protein
+standards with fixed concentrations made and absorbance measured for generating
+standard curve (concentration vs absorbance). </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Conducted Western blot </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>SDS-Page gel set up with purified
+proteins, cyanobacterial lysate and <span class=SpellE>ecoli</span> vector only
+lysate (not used in this specific assay). Proteins purified and loaded on
+-20% gel in 2 fold <span class=SpellE>diluations</span> from 100 ng to 1.56
+ng. Proteins loaded with 3 x loading dye and denatured at 70C for 10 mins.
+Empty lanes loaded with 1 x loading dye. Gel run at 300 v for 22 mins. </span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Transfer constructed with membrane
+sandwich -plastic latched case, two <span class=SpellE>whatman</span> papers,
+two sponges, membrane, <span class=GramE>gel</span>. Transfer apparatus run for
+.5 hours at 90 V. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ran another PCR of MC002_b1, MC003_b1</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Ran PCR of gel extracted blocks
+MC002_b1 and MC003_b1</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/12 </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Blocking and Staining</span></i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span class=SpellE><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:Arial;color:black'>Ponceau</span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:Arial;
+color:black'> staining conducted -membranes incubated in <span class=SpellE>ponceau</span>
+reagent for 5 mins. Could see faint bands for <span class=SpellE>KaiA</span>
+and <span class=SpellE>KaiC</span> samples. Membrane pieces blocked with 2% <span
+class=SpellE>milkd</span> and TBST overnight. </span><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/13 </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>No primary antibody available at time,
+continued to block. </span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/14 </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Membrane pieces stained with primary
+Kai rabbit antibodies for 1.5 hours. Membrane washed with washing buffer for 10
+mins for 3 times (30 mins total). Membrane stained with secondary goat antibody
+for 1 hour. Membrane pieces washed with washing buffer for 10 mins for 3 times
+(30 mins total). Membrane imaged. Only kai B showed up in image. &nbsp;</span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>9/15</span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Induction</span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-bidi-font-family:Arial;color:black'>Induction repeated. Cells pelleted,
+washed with 1 mL water, pelleted then reintroduced into <span class=SpellE>Lb</span>
+or M9 media. Concentrations:</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman";
+mso-bidi-font-family:"Times New Roman"'><br style='mso-special-character:line-break'>
+<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
+<![endif]><o:p></o:p></span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse;mso-yfti-tbllook:1184'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
+  solid black .25pt;mso-border-alt:solid black .25pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
+  border-right:solid black 1.0pt;mso-border-top-alt:solid black .25pt;
+  mso-border-left-alt:solid black .25pt;mso-border-alt:solid black .25pt;
+  padding:5.25pt 5.25pt 5.25pt 5.25pt'></td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p>&nbsp;</o:p></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><o:p>&nbsp;</o:p></span></p>
+</div>
+</body>
 </html>
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