Difference between revisions of "Team:UChicago/Notebook"

Latest revision as of 07:49, 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.

Although this as much data as we have as of a few days before the Wiki freeze, we will definitely have important updates to come we hope to present during the Jamboree.

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+	{margin-bottom:0in;}
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+	{margin-bottom:0in;}
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+</style>
-<p> Document the dates you worked on your project.</p>
+</head>
-<h5>What should this page have?</h5>
+<body lang=EN-US link=blue vlink=purple>
-<ul>
-<li>Chronological notes of what your team is doing.</li>
+<div class=WordSection1>
-<li> Brief descriptions of daily important events.</li>
-<li>Pictures of your progress. </li>
+<p class=MsoNormal align=center style='text-align:center'><b><span
-<li>Mention who participated in what task.</li>
+style='font-size:26.0pt;font-family:"Calibri",sans-serif;color:#C00000'>UChicago
-</ul>
+Summer 2015 Lab Journal</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><i><span
+style='font-family:"Arial",sans-serif'>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.</span></i></p>
+<p class=MsoNormal align=center style='text-align:center'><i><span
+style='font-family:"Arial",sans-serif'>--</span></i> <i><span style='font-family:
+"Arial",sans-serif'>Eduard Christoff Philippe Gerard Renaldi, Prince of Genovia,
+The Princess Diaries</span></i></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>&nbsp;</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Week 1: </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Goals- take inventory, design constructs and primers, test
+competent cells</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/15/15 </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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;color:#1155CC'>http://advances.sciencemag.org/content/1/5/e1500358.full</span></a></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Took inventory, created excel sheet online under <u>Protocols
+folder “Genehackers 2015 Inventory”</u></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Downloaded SnapGene Viewer to work on plasmid constructs. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/16/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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:</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Read-out activator (KaiCEE-RFP, RpaA, SasA) </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Test gene (kaibc promoter, GFP)</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Fusion SasA</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Fusion KaiC-P </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Last two based heavily on paper (Chen, 2015)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Met with Jennifer Moran -need to do safety training before
+autoclaving liquids and other procedures, will accomplish once more people are
+back.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#9900FF'>To Discuss: Is it worth having negative feedback regulator of
+RpaA/ output molecule?</span></i></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/17/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decided to use CikA as negative regulator/inhibitor instead of Lab
+A. CikA better characterized. Reviewed (Gutu. O’Shea, 2013). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/18/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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 kaibc/Kai analog sequences. Decided on meeting 4pm Tuesday. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#9900FF'>To Discuss: Specific RBS and promoter strengths on different
+genes. </span></i></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#4CBF26'>Started Testing for Competent Cells</span></u></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>*Used Justin/Rust Lab protocol instead of iGEM/team protocol
+because did not have cmrR plates. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<h4>Inspiration</h4>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>You can see what others teams have done to organize their notes:</p>
+color:black'>Labels:</span></p>
-<ul>
+<ul style='margin-top:0in' type=disc>
-<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
-<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+     style='font-size:16.0pt;font-family:"Garamond",serif'>Plasmid of Interest-
-<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+     PJ006, containing KaiABC under kaiA, and kaibc promoters, Spec resistance,
-<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+     Conc= 100ng/uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>Transformed plate-
+     PJ006 MC 6/18/15 </span></li>
 </ul>
-<!-- autogen start -->
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Steps</span></p>
-<p><b>GeneHackers Summer 2015 Journal</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Remove cells from freezer, incubate on ice </span></p>
-</html>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-Original [[File:UChicagoLabNotebook.pdf]]
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<html>
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 1 uL DNA (100ng/uL) into competent cell tube </span></p>
-<p><b>Week 1: </b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<p><b>Goals- take inventory, design constructs and primers, test competent
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-cells</b></p>
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube on ice for 30 mins</span></p>
-<p>6/15/15 </p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Took inventory, created excel sheet online under <u>Protocols folder =E2=80=9CGenehackers 2015 Inventory=E2=80=9D</u></p>
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
-<p>Downloaded SnapGene Viewer to work on plasmid constructs. </p>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube 42C water bath for 1 min heat shock</span></p>
-<p>6/16/15</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<ol><li>Read-out activator (KaiCEE-RFP, RpaA, SasA) </li>
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
-<li>Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)</li>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Test gene (kaibc promoter, GFP)</li>
+color:black'>Incubate tube ice 5 mins </span></p>
-<li>Fusion SasA</li>
-<li>Fusion KaiC-P </li></ol>
-<p>Last two based heavily on paper (Chen, 2015)</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</span></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 style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>*Used LB from MJR Lab, will have to
+make LB tomorrow </span></p>
-<p><b>To Discuss: Is it worth having negative feedback regulator of RpaA/ output molecule?</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube in shaker 37C for 1 hour</span></p>
-<p>6/17/15</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
-<p>Decided to use CikA as negative regulator/inhibitor instead of Lab A. CikA better characterized. Reviewed (Gutu. O=E2=80=99Shea, 2013). </p>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Heat Spec plate in incubator as cold plate reduces
+efficiency,complete while cells shaking</span></p>
-<p>6/18/15</p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><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;color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Collect pellet, spin 3000 rcf/gs for 3 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>10.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decant 800 uL of supernatant </span></p>
-<p><b>To Discuss: Specific RBS and promoter strengths on different genes. </b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>11.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use glass beads (5-6) per section (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>12.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mix pellet, pipetted 200 uL in total, 180 to 0.90 section, 20 to
+.10 section </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>13.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Shake with beads and remove </span></p>
-<p><b>Started Testing for Competent Cells</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><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;color:black'>14.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate plate overnight 37C</span></p>
-<p>Labels:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<ul class=3D"small"><li>Plasmid of Interest- PJ006, containing KaiABC under kaiA, and kaibc promoters, Spec resistance, Conc=3D 100ng/uL</li></ul>
-<li>Transformed plate- PJ006 MC 6/18/15 </li></ul>
-<p>Steps</p>
-<ol><li>Remove cells from freezer, incubate on ice </li>
-<li>Add 1 uL DNA (100ng/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 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 align=center style='text-align:center'><span
-<p><b>Checked on Transformed Plates</b></p>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/19/15</span></p>
-<p>Good growth on both sections. </p>
-<p>Transformation efficiency</p>
-<p><blockquote>0.10 Section</blockquote></p>
-<p>      =3D (293 cfus) / ( ((1 uL x 100 ng/uL)/1000 uL soln)(20/200 uL plated))</p>
-<p>		      =3D 293 cfus/0.01 ng DNA plated =3D <u>2.93 x 10^4 transformants/ng</u></p>
-<p><b>To Discuss: How to improve Transformation Efficiency. </b></p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#CC0000'>Checked on Transformed Plates</span></u></b></p>
-<p><b>Made 500 mL LB Solution </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Good growth on both sections. </span></p>
-<p><b>Made CM plates</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	</p>
+color:black'>Transformation efficiency</span></p>
-<p>Temperature of Freezer=3D 6 C, Chloramphenicol storage temperature=3D 2-8 C </p>
-<p>125 uL of 50mg/ml cm used for every 250 uL plate soln made </p>
-<p>	Poured plates</p>
-<p>	Need to label once plates set overnight</p>
-<p>Updated restock list, will go tomorrow to buy new supplies (check Inventory) </p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>0.10 Section</span></i></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;= (293
+cfus) / ( ((1 uL x 100 ng/uL)/1000 uL soln)(20/200 uL plated))</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>
+color:black'>                   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;= 293 cfus/0.01
-<p>Placed cmR plates in left cold room, bottom right corner of room behind some Rust plates </p>
+ng DNA plated = <u>2.93 x 10^4 transformants/ng</u></span></p>
-<p><b>Week 2: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Goals- Finalize construct design, develop Competent Cells</b></p>
-<p>						6/22/15</p>
+<p class=MsoNormal><b><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Worked on Week 1 Presentation</p>
+color:#9900FF'>To Discuss: How to improve Transformation Efficiency. </span></i></b></p>
-<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>
-<p>Started Mini-prep to extract Kai proteins. Inoculated 2 colonies, 1 each in 2mL LB + Spec medium. </p>
-<p>6/23/15 </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Cultured 5 colonies from LB only plate into 10 mL LB </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><u><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>
+color:#4CBF26'>Made 500 mL LB Solution </span></u></b></p>
-<p>6/25/15 </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p>6/26/15 </p>
+<p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#4CBF26'>Made CM plates</span></u></b></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>
+color:black'>          </span></p>
-<p>https://drive.google.com/open?id=3D0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV FIX THIS LINK</p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>Temperature of Freezer= 6 C,
+Chloramphenicol storage temperature= 2-8 C </span></p>
-<p>Used both Rust Lab and RbCl2 as a comparison. </p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>125 uL of 50mg/ml cm used for every
+uL plate soln made </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>
+color:black'>          Poured plates</span></p>
-<p>6/29/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Conducted Transformation of CaCl2 and RbCl2. </p>
+color:black'>          Need to label once plates set overnight</span></p>
-<li>Remove cells from freezer, incubate tubes on ice </li>
-<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><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>		      =3D 52 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>1.15 x 10^6 transformants/ng (Rust)</u></p>
+color:black'>Updated restock list, will go tomorrow to buy new supplies (check
-<p>=3D52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 uL plated))</p>
+Inventory) </span></p>
-<p>		      =3D 18 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>4.00 x 10^5 transformants/ng (RbCl2)</u></p>
-<p>6/30/15</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</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 align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/20/15</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>
+color:black'>Stock room closed :(, Will order things on Monday</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>
+color:black'>Placed cmR plates in left cold room, bottom right corner of room
+behind some Rust plates </span></p>
-<p>7/21/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>gBlocks for Oscillation and Read-Out systems were finally ordered. Primers were designed. </p>
-<p>7/22/15</p>
+<p class=MsoNormal><b><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>
+color:#1155CC'>Week 2: </span></b></p>
-<p>GFP Protocols</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:#1155CC'>Goals- Finalize construct design, develop Competent Cells</span></b></p>
-<p>7/23</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Materials for practice western blot acquired.  Gibson Assembly protocol drafted. </p>
-<p>7/24</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>                                                          6/22/15</span></p>
-<p>7/27</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>Worked on Week 1 Presentation</span></p>
-<p><b>Week 5: </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- Finalize and outline protocols, generate explanations for plasmids and background info for wiki and presentation, order materials, gblock assembly</b></p>
+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 E.Coli
+/21/15”</span></p>
-<p>7/28</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>Started Mini-prep to extract Kai proteins. Inoculated 2 colonies,
+each in 2mL LB + Spec medium. </span></p>
-<p>7/29</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p>7/30/15</p>
+<p class=MsoNormal align=center style='text-align:center'><span
-<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>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/23/15 </span></p>
-<p>7/31/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gibson Assembly</b></p>
+color:black'>Cultured 5 colonies from LB only plate into 10 mL LB </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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Week 1 meeting today</span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Meeting Notes:</span></p>
-[[File:NotebookTable1.png]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&#8594;</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> 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. </span></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&#8594;</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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
+KaiABC as submitted biobrick so perhaps focusing on biosynthesis pathway is a
+bit ambitious. Need to consider perhaps alternative, simpler molecule. </span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>&#8594;</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Recipe Phusion Master Mix:</p>
+color:black'> Will likely assemble using Gibson, order this free kit from RPI. </span></p>
-<p>o   Phusion 5X GC Buffer =3D 10uL x 5.5 =3D 55 uL</p>
-<p>o   dNTPs 10 mM  =3D 1 x 5.5 =3D5.5 uL</p>
-<p>o   F Primer MC003 10 uM =3D 2.5 x 5.5 =3D13.75 uL</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Goals- gblock assembly and Transformation</b></p>
+color:black'>Contact Danny for competent cells and Justin for Gibson primers</span></p>
-<p>8/3/15</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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;color:black'>6/24/15 </span></p>
-<p>Readout SasA MC002 =E2=80=93Amp (theoretically want to use with MC001 if successful) =C3=A0 need to add GFP + kai bc</p>
-<p>            	Readout SasA/CikA MC003 =E2=80=93Amp (=E2=80=9C  =E2=80=9C) =C3=A0 need to add GFP + kaibc</p>
-<p> 		KaiC Variants MC004-7- Cmr (would never use with MC001)</p>
-<p><b>Agarose Gel Casting</b></p>
+<p class=MsoNormal><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>
+color:black'>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. </span></p>
-<p><b>Gel Electrophoresis</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p><b>Image:</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/25/15 </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/26/15 </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Carried out competent cell procedure. </span></p>
+<p class=MsoNormal><a
+href="http://www.unc.edu/depts/marzluff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gel Extraction</b></p>
+<p class=MsoNormal><a
+href="https://drive.google.com/open?id=0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#1155CC'>https://drive.google.com/open?id=0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxPU01uclJCVmNV</span></a></p>
-<p>Gel Weights:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>MC001 =E2=80=93N/A</p>
-<p>MC004 -0.1536 g</p>
-<p>MC005 -0.2048 g</p>
-<p>MC006 -0.0965 g</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>
+color:black'>Used both Rust Lab and RbCl2 as a comparison. </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><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gel Electrophoresis</b></p>
-<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>
-<p><b>Image:</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Week 3: </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Goals- Test efficiency of competent cells, start as much cloning
+as possible</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/29/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Conducted Transformation of CaCl2 and RbCl2. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>15.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Remove cells from freezer, incubate tubes on ice </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>16.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 1 uL DNA (50 pg//uL) into competent cell tube </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>17.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube on ice for 30 mins</span></p>
-<p><b>Gibson Assembly</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
-<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>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>18.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube 42C water bath for 1 min heat shock</span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
-<p>Master mix of 70 uL created (calculate ratio of 1X x 7/5)</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>19.<span
-<p>o   Phusion 5X HC Buffer =3D 14 uL</p>
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p>o   dNTPs 10 mM  =3D 1.4  uL</p>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   Phusion DNAP =3D 0.7 uL</p>
+color:black'>Incubate tube ice 5 mins </span></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 style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
-<p>Gel Electrophoresis</p>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>20.<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:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p><b>Image:</b></p>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rescue cells by pipetting 850 uL LB into tube (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>21.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube in shaker 37C for 1 hour</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>22.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Heat Cam plate in incubator as cold plate reduces
+efficiency,complete while cells shaking</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>23.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Collect pellet, spin 3000 rcf/gs for 3 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>24.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decant 800 uL of supernatant </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>25.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use glass beads (5-6) per section (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>26.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mix pellet, pipetted 200 uL in total</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>27.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Shake with beads and remove </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>28.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate plate overnight 37C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spec on LB negative control culture overnight =-0.019 A (no growth
+at all)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gel Extraction</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>Used Promega spin columns/buffer to concentrate in 15 uL of DNA </p>
+font-family:"Garamond",serif;color:black'>= (52 cfus) / ( ((1 uL x 50 pg/uL x
+ng/1000pg)/1000uL soln))*((180/200 uL plated))</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>
+color:black'>                   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;= 52 cfus/(4.5 x 10^-5)
-<p>4 - 34.7</p>
+ng DNA plated =<u>1.15 x 10^6 transformants/ng (Rust)</u></span></p>
-<p>4=E2=80=99 -</p>
-<p>5 - 31.5</p>
-<p>6 - 36.3</p>
-<p>7 - 40.1</p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p><b> </b></p>
+font-family:"Garamond",serif;color:black'>=52 cfus) / ( ((1 uL x 50 pg/uL x
-<p><b>PCR</b></p>
+ng/1000pg)/1000uL soln))*((180/200 uL plated))</span></p>
-<p>To redo PCR for Gibson products of MC001, 75 uL of 1X PCR Master Mix</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>
+color:black'>                   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;= 18 cfus/(4.5 x
+^-5) ng DNA plated =<u>4.00 x 10^5 transformants/ng (RbCl2)</u></span></p>
-<p><b>8/6/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gel Electrophoresis</b></p>
-<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>
-<p><b>Image:</b></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>6/30/15</span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-[[File:UChicagoGel1.png]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b> </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gibson Assembly:</b></p>
+color:#1155CC'>Week 4: </span></b></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;
+color:#1155CC'>Goals- Order final gBlocks and primers. Standardize and develop
+specific, in depth protocols. Practice Western Blots, start writing project
+report. </span></b></p>
-[[File:UChicagoTable2.png]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<html>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>7/20/15</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>
+color:black'>gBlocks for Oscillation and Read-Out systems were modified. See
+Dropbox for final edits and modifications. </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'>&nbsp;</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 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 align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>7/21/15</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;
+color:black'>gBlocks for Oscillation and Read-Out systems were finally ordered.
+Primers were designed. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal align=center style='text-align:center'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>7/22/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>GFP Protocols</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;color:#1155CC'>http://advances.sciencemag.org/content/advances/1/5/e1500358.full.pdf</span></a></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7/23</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Materials for practice western blot acquired. &nbsp;Gibson
+Assembly protocol drafted. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>7/24</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>
+color:black'>Started western blot. See Rust Lab protocol. Slight changes
-<p>o   dNTPs 10 mM  =3D 1 uL</p>
+include 7.5% gel used, cassette assembled not submerged in buffer. Primers
-<p>o   Phusion DNAP =3D 0.5 uL</p>
+diluted and placed in -20 fridge. </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'>&nbsp;</span></p>
-<p>Gibson 8/3 -MC003/MC004</p>
-<p>Gibson 8/4 -MC003/MC004</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;
+color:black'>7/27</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>
+color:black'>Primary and secondary antibody staining accomplished. Experiments
-<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>
+more clearly laid out. Need to start considering plan for pRha inducible
+promoter and how to alter stoichiometry.</span></p>
-<p><b>Image:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Week 5: </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Goals- Finalize and outline protocols, generate explanations for
+plasmids and background info for wiki and presentation, order materials, gblock
+assembly</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7/28</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7/29</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Finished specific protocols -need to ask White lab for sonicator?.
+Looked up compatibility of backbones. pSB1,3,4 have pMB1 (copy number
+-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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7/30/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Materials reviewed and listed. Meeting with Barry to talk about
+iGEM as a class. Went over protocols and methods. Allocated who is ordering
+what.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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>
+color:black'>7/31/15</span></p>
-<p>Plate Numbers are indicated on lower left of the pictures.</p>
-<p>Average is 321 colonies.</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly</span></b></p>
-<html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-[[File:UChicagoPlate1_1.jpg]]
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-[[File:UChicagoPlate1_2.jpg]]
+color:black'>5 uL of Gibson HiFi Master mix was used in each assembly reaction
-[[File:UChicagoPlate1_3.jpg]]
+to minimize amount reagent used. Amount of blocks used, dependent on bps of
-[[File:UChicagoPlate1_4.jpg]]
+each block relative to each other. Each gblock diluted in 20 uL 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 heatblock for 1 hour.</span></p>
-</html>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Transformation Efficiency </b></p>
+<p class=MsoNormal><img width=1021 height=285 src="..//wiki/images/3/3b/UChicago2015Notebook_filesimage001.jpg"
+align=left hspace=12 alt="mage 1.png"></p>
-<p>Used 1ul of 50pg/ul  of DNA </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>4: 287 / 5*10^-5 =3D 5.74*10^6 cfu/ug</p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>5: 328 / 5*10^-5 =3D 6.56*10^6 cfu/ug</p>
+color:black'>PCR</span></b></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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 dH<sub>2</sub>O.
+Aliquoted 49 uL of Master Mix with 1 uL from Gibson Assembly Mix.</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>
+color:black'>Recipe Phusion Master Mix:</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>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X GC Buffer = 10uL x 5.5 = 55 uL</span></p>
-<p>Ingredients:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	Q5 High-Fidelity 2X Master Mix- 25 uL</p>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	DNA -1 uL</p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<p>	F Primer -2.5 uL</p>
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 1 x 5.5 =5.5 uL</span></p>
-<p>	R Primer -2.5 uL</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>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC003 10 uM = 2.5 x 5.5 =13.75 uL</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC004 10 uM = 2.5 x 5.5= 13.75 uL</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>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>5 uL 10X buffer</p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<p>1 uL dNTPs</p>
+"Garamond",serif;color:black'>Phusion DNAP = 0.5 x 5.5 =2.75 uL</span></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;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 32.5 x 178.75 uL</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;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 1.5 x 5.5 =8.25 uL</span></p>
-<p><b>8/9/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>*Should have added only 170.5 uL (31 x 5.5) &nbsp;–Mix slightly
+more dilute</span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal><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>
+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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Week 6: </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#1155CC'>Goals- gblock assembly and Transformation</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/3/15</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decided on backbones:</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;        </span></b><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>Oscillator
+MC001 – Cmr (standard igem backbone for submitted biobrick)</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>Readout SasA MC002 –Amp
+(theoretically want to use with MC001 if successful) à need to add GFP + kai bc</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;        Readout
+SasA/CikA MC003 –Amp (“ &nbsp;“) à need to add GFP + kaibc</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>                   KaiC Variants MC004-7- Cmr (would never use
+with MC001)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Agarose Gel Casting</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Made 50 mL of 1% Agarose gel. General procedure: added agarose and
+X 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
+-20 mins.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>8/10/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image:</span></b></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>Gel repeated, this time using leftover 45uL of sample. 1kB Plus invitrogen ladder used. </p>
+font-family:"Garamond",serif'><img border=0 width=1060 height=1182
+id="Picture 2" src="..//wiki/images/c/ca/UChicago2015Notebook_filesimage002.png"
+alt="https://lh4.googleusercontent.com/FMG8G6gOA0iIJ6_IhKuvzNmIV7rdLH3kRSesuFoQ7q97DhYI3f_adrE941N4k51vngEbvrE2aNe30xKGMH1Doj5JA4EztaYeNF3RA_5EXpfZum143oJciRXDxW_QwY1owUI23-4"></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+</span><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Extraction</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Weights:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC001 –N/A</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC004 -0.1536 g</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC005 -0.2048 g</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC006 -0.0965 g</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC007 -0.3962 g</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>*1mg = 1uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Added 3x uL QG buffer to volume of gel and 1X uL of isopropanol to
+volume of gel.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>To redo PCR for MC001,4,5,6,7, 1X Master Mix for 15 reactions
+made. Two reactions for each construct.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Master Mix Recipe-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X GC Buffer = 150 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 15 &nbsp;uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 7.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 465 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 22.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Added 2.5uL of F and R Primers, 44 uL of Master Mix, &nbsp;and 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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Master mix also used for amplifying linear Cmr backbones
+(diluted in 10uL, used 1 uL of sample for Phusion PCR).</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/4/15</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span></p>
-<p><b>Innoculation-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p><b>Gibson Assembly-</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Gibson assembly of BH001_b1 and Cam backbone conducted. </p>
+color:black'>Image:</span></b></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Set up PCR for biobricks MC002, and MC003 and BH001 (confirmation).  </p>
-<p>11X PCR Master Mix</p>
-<p>PCR Master Mix Recipe-</p>
-<p>o   Phusion 5X HF Buffer =3D 110 uL</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'><img border=0 width=940 height=897 id="Picture 3"
+src="..//wiki/images/2/20/UChicago2015Notebook_filesimage003.png"
+alt="https://lh4.googleusercontent.com/vhr9tI0AP8GjCG1NHKQ1TBox7RaB6XEsB5IFctg6cGxmBqjaYWc2jYikx9hAZOvmlMtz2s1ksbRWM0hC6OUiM2y2kx-8zU3SFT_Edk-ZqvzjOpMiR0sHZ0xY_Y75RXMdAiL2nrg"></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly</span></b></p>
-<p><b>DNA Template</b></p><p><b>Primers</b></p><p><b>Info</b></p><p><b>Gel to Run</b></p><p>MC002_b1</p><p>MC029, MC010</p><p>1815 bps</p><p>Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts</p><p>1% Agarose</p><p>MC003_b1</p><p>MC029, MC014</p><p>1815 bps</p><p>Clone out b1 to give right initial sequence for kaibc/GFP/Term inserts</p><p>1% Agarose</p><p>MC008_b1</p><p>MC003, MC028</p><p>1294 bps</p><p>Clone out kaibc promoter/GFP</p><p>1% Agarose</p><p>Biobrick B0015 Terminator</p><p>MC027, MC030</p><p>129 bps</p><p>Clone out terminator</p><p>2% Agarose</p><p>BH001/Cam  Gibson</p><p>MC003, MC004</p><p>765 bps</p><p>*should not have done</p><p>? Was to clone out insert, should have transformed as is. </p><p>1% Agarose</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5 uL of Gibson HiFi Master mix, 1 uL PMC001_b1, 0.5965 uL
+PMC001_b2, 3.4305 uL H<sub>2</sub>O, heatblock for 1 hour 50<sup>o</sup>C.</span></p>
-<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'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Master mix of 70 uL created (calculate ratio of 1X x 7/5)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HC Buffer = 14 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 1.4 &nbsp;uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 0.7 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 43.4 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 2.1 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA =1.4 uL</span></p>
-<p><b>8/11/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC003= 3.5 uL</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>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC004 =3.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>10uL Master mix aliquoted into 7 samples.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup> 15 s,
+<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>, 66<sup>o</sup>, 68<sup>o</sup>,
+<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10
+min, 4<sup>o</sup> hold.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>, 62.0<sup>o</sup>,
+.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>, 72.0<sup>o</sup></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup> for 30 cycles</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/5/15</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image:</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<img border=0 width=1014 height=822 id="Picture 4"
+src="..//wiki/images/9/9f/UChicago2015Notebook_filesimage004.png"
+alt="https://lh5.googleusercontent.com/ep3k5EhBnMexsc-fQ-22FFqWJRGb3vGSet4vO6KpuOEVcFUzp2DGnVNpFkpcQpiX10kcAqz_7IgsH0qTP76UXscOgzGidyp2zjqUFnsAbz5Ql6aApcrL0bBNjreWJP6HAfeNEwg"><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Extraction</span></b></p>
-<p><b>Miniprep</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>BH protocol</b></p>
+color:black'>Used Promega spin columns/buffer to concentrate in 15 uL of DNA </span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>	</b><u>PCR of Minipreps</u></p>
-<p>(BH protocol)</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>
+color:black;background:lime'>Purity Yields using nanodrop - C1 (Cam Backbone) -</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;
+color:black'>C2 (Cam Backbone) -58.9 ng/uL</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4 - 34.7</span></p>
-<p>Thermocycler Settings-</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>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>
+color:black'>4’ -</span></p>
-<p>Thermocycler Settings for Mini-Prep PCR-</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>
+color:black'>5 - 31.5</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6 - 36.3</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black;background:lime'>7 - 40.1</span></p>
-<p><b>8/12/15</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
-<p>PCR of Terminator </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>To redo PCR for Gibson products of MC001, 75 uL of 1X PCR Master
+Mix</span></p>
-<p>2 50 uL Samples  </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Master Mix Recipe-</span></p>
-<p>	HF Buffer -10 uL</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	DMSO -1.5 uL</p>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	DNTPs -1 uL </p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<p>	H2O -31 uL</p>
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 15 uL</span></p>
-<p>	Phusion DNAP -0.5 uL</p>
-<p>	10 uM of MC027 Primer -2.5 uL </p>
-<p>	10 uM MC030 Primer -2.5 uL</p>
-<p>	DNA from plate -1 uL</p>
-<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;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 15 &nbsp;uL</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. 1kB plus Invitrogen ladder used. Send samples 41,43,51,52,53,54,61,62,63,64,74 for sequencing. </p>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = .75 uL</span></p>
-<p><b>Images:</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 46.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 2.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA (products from Gibson 8/4)= 1.5uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC003= 3.75uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC004 =3.75uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>10 uL of Master Mix aliquoted into each sample tube.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>To PCR PMC001_b1 for confirmation of block and analysis of primers</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>50 uL of 1XPCR Master Mix Recipe-</span></p>
-<p><b>Gel Purification-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Extracted correctly sized product fragmnets under blue light: T-189 bps, GFP 1249 bps. </p>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Weights of gels:</p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<p>GFP 1-63.8 mg</p>
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 10 uL</span></p>
-<p>GFP 2-70.64 mg</p>
-<p>T 1-34.6 mg</p>
-<p>T 2-110.0 mg</p>
-<p>Used Machery-Nagel clean up method:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<ol><li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Incubate at 50C for 10 mins</li>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<li>Transfer solution to spin column and collection tube. </li>
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 1 &nbsp;uL</span></p>
-<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;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = .5 uL</span></p>
-<p>Purity recorded w/Nanodrop:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>T1- 5.1ng/uL 260/280-14.84</p>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>T2-23.1ng/uL 260/280-1.92</p>
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
-<p>GFP 1- 41.6ng/uL 260/280-1.94</p>
+"Garamond",serif;color:black'>H<sub>2</sub>O= 31 uL</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;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 1.5 uL</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA (pMC001_b1)= 1 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC005= 2.5uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC006 =2.5uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Added 2.5uL of F and R Primers, 44 uL of Master Mix, &nbsp;and 1uL
+of DNA for each sample..</span></p>
-<p><b>Mini-Prep Results (Nano-Drop)</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup> 15 s,
+<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>, 66<sup>o</sup>, 68<sup>o</sup>,
+<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10
+min, 4<sup>o</sup> hold.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>, 62.0<sup>o</sup>,
+.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>, 72.0<sup>o</sup></span></p>
-<p>The highlighted ones are the ones that we choose to sequence.</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup> for 30 cycles</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/6/15</span></b></p>
-<p><b>Sequencing-</b></p>
+<p class=MsoNormal><b><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>
+color:black'>Gel Electrophoresis</span></b></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><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>MC003 (F) [2]</p>
+color:black'>Made 100 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading
-<p>MC041 (F) [3]</p>
+dye) and 1 50uL sample (divided into two wells, 42uL in one well 18 in the
-<p>MC023 (F) [4]</p>
+other). Run for 120V, 30 mins. No product clear enough to extract. Imaging gel
-<p>MC022 (R) [5]</p>
+shows faint products under 68,70, and 72<sup>o</sup>. Could mean issue with
-<p>VR [6]</p>
+primers. Strangely, no product of right gBlock size seen. Again could be
-<p>[41]</p>
+primers.</span></p>
-<p>[43]</p>
-<p>[53]</p>
-<p>[54]</p>
-<p>[62]</p>
-<p>[63]</p>
-<p>[71]</p>
-<p>[73]</p>
-<p><img src=3D"cid:Image_7.png" /></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>^Things sent.</p>
-<p><b>Transformation of BH001:</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>
+color:black'>Image:</span></b></p>
-<li>Remove cells from freezer, incubate tubes on ice </li>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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 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><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=629 height=509 id="Picture 5"
+src="..//wiki/images/9/99/UChicago2015Notebook_filesimage005.jpg" alt="igure 4.png"></span></b></p>
-<p><b>8/13/15-</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly:</span></b></p>
-<p><img src=3D"cid:Image_8.png" /></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Transformation Efficiency of BH001-</p>
+color:black'>Assembled purified biobricks MC004, MC005, MC006, MC007 into Cam
-<p>amt dna used/plate</p>
+backbone. Used following recipe based on bps of insert and backbone. </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;
+color:black'><img border=0 width=1194 height=161 id="Picture 6"
+src="..//wiki/images/7/74/UChicago2015Notebook_filesimage006.png"
+alt="https://lh3.googleusercontent.com/zyijYOnOqRFCBDdEEMBCRewGmCzJVhakw5hLpdUOsgGPeQARFugu30Vc4sBXfhFJ-WA0uuAX13-2qz6DuMIy-dsK0BJTfKWl1SanXnyWPnYCab_E-wUmAR3z0oDKEkclhEaFrM0"></span></b></p>
-<p>They were PCRed separately with two different annealing temperatures, 61.6 and 66. </p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gibson Assembly</b></p>
+<p class=MsoNormal><b><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>
+color:black'>Transformation of Assembled products:</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>DNA straight from Gibson Assembly Reaction was transformed into
+competent cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x
+^5 transformants/ng (RbCl2) </u></span></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Prepared 2 50uL 1X PCR samples. </p>
-<p>	HF Buffer -10 uL</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 style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Remove cells from freezer, incubate tubes on ice </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 style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 1 uL DNA (50 pg//uL) into competent cell tube </span></p>
-<p>Thermocycler Settings for Mini-Prep PCR- Low Temp</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube on ice for 30 mins</span></p>
-<p>Thermocycler Settings for Mini-Prep PCR- High Temp</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube 42C water bath for 1 min heat shock</span></p>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube ice 5 mins </span></p>
-<p><b>Image-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube in shaker at 37C/1100 RPM for 1 hour</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Heat Cam plate in incubator as cold plate reduces
+efficiency,complete while cells shaking</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Collect pellet, spin 3000 rcf/gs for 3 mins </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decant 800 uL of supernatant </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use glass beads (5-6) per section (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mix pellet, pipetted 200 uL in total</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Shake with beads and remove </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate plate overnight 37C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<img border=0 width=1394 height=1323 id="Picture 7"
+src="..//wiki/images/4/4a/UChicago2015Notebook_filesimage007.png"
+alt="https://lh3.googleusercontent.com/s_QBM9SKfAUn9wjfvTWl9K00NTt1kxDu5VMMaLhIMAGa_yQ1-Wauhe7DvycjA8ELKmiv1cnttv3XJOhb_79JO_NisWwYJr9KmP_U6VxF8cb5wChdZvi1yCBwYSUFd84SuQP0jSE"></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>*Negative control w/no transformed DNA resulted in 0 colonies.
+Negative control set up on 8/7.</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR of 8/3 Gibson PCR, 8/4 Gibson PCR conducted for further
+amplification. Block 4.1 PCR as positive control. Block 1.1 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). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Added Ingredients to individual Samples</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HC Buffer = 10 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 1 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 0.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 31.0 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 1.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA =1.0 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer = 2.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer =2.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>10uL Master mix aliquoted into 7 samples.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup> 15 s,
+<sup>o</sup>, 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10 min, 4<sup>o</sup>
+hold.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Gel Extraction-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>BH003 biobrick extracted from gel. Machery-Nagel protocol used for gel extraction. </p>
+color:black'>Primers for each sample</span></p>
-<p>Weight of gel: 85.2 mg</p>
-<p>Used Machery-Nagel clean up method:</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+color:black'>Gibson 8/3 -MC003/MC004</span></p>
-<li>Incubate at 50C for 10 mins</li>
-<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>Nanodrop purity- 14.4 ng/uL, 260/280- 1.55</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson 8/4 -MC003/MC004</span></p>
-<p><b>PCR-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>pMC001_b1 -MC005/MC006</span></p>
-<p>PCR Master Mix Recipe-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   Phusion 5X HF Buffer =3D 50 uL</p>
+color:black'>pMC001_b2 -MC007/MC008</span></p>
-<p>o   dNTPs 10 mM  =3D 5  uL</p>
-<p>o   Phusion DNAP =3D 2.5 uL</p>
-<p>o   H2O =3D 155 uL</p>
-<p>o   DMSO =3D 7.5 uL</p>
-<p>o   DNA (products from Gibson 8/4)=3D 1.0uL</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>pMC004_b1- MC019/MC020</span></p>
-<p><b>Gibson Assembly-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p><img src=3D"cid:Image_9.png" /></p>
-<p>	</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>
+color:black'>*Upon further examination, should have used MC003 for pMC004_b1</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Image- </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/7/15</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image:</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><img border=0 width=649 height=569 id="Picture 8"
+src="..//wiki/images/6/62/UChicago2015Notebook_filesimage008.png"
+alt="https://lh3.googleusercontent.com/UOM5sBy_cJQOet0PiJmfpedxpicfly2pJkyIUvzu2-y4wXcmcG_PNCPQRkd9Koo1GU0HKO4YDoQl-NjdOAOtmLuL8fs1i3gi6hF-ertbgQfEfevF35uUmuaNW4zknTg8olL87us"></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation Results</span></b><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'> </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Used iPhone application “ColonyCount” to assist in counting
+plates.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Plate Numbers are indicated on lower left of the pictures.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Average is 321 colonies.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=720 height=1280 id="Picture 9"
+src="..//wiki/images/8/8d/UChicago2015Notebook_filesimage009.jpg"
+alt="https://lh3.googleusercontent.com/X-VD4nUUhpipdr0rfrWJOvsNhjSK970Lyeqg4-7_LgaPEpds_4wfL591u_Zrf_LUL8d8TvSHd1p6C84HMcW1AoGnZqSuSnFUe2crNaJNiycNdr9dwgobKH7qmEoP5OK-XhGzmyc"><img
+border=0 width=720 height=1280 id="Picture 10" src="..//wiki/images/f/f2/UChicago2015Notebook_filesimage010.jpg"
+alt="https://lh5.googleusercontent.com/PesRqsJVYxBn1kZnwLvigIlmVTqnti6nGnaTtqg69mfieJDULTGYc7yy7sG9sQowMvk-UtSCSGPpjLWHZhoiXs4FvtwjF2us2qEZsBPZX-VBmFiKuyqNNGho_jZxns73zKP9NfI"><img
+border=0 width=720 height=1280 id="Picture 11" src="..//wiki/images/8/8e/UChicago2015Notebook_filesimage011.jpg"
+alt="https://lh6.googleusercontent.com/f1PFy4vU3TSCHwvIvj6Hf9GOz07r3Ubh6I_2a7lyP9V_f4q8nCrF_pKdBp81mYTjtbj8DH4r1_lrauKjIWCEBG2EKvcJEqgN8a4Yill_iDPPCoRRRXiawIsUR4-tkbbZlIiFYsY"><img
+border=0 width=720 height=1280 id="Picture 12" src="..//wiki/images/8/88/UChicago2015Notebook_filesimage012.jpg"
+alt="https://lh5.googleusercontent.com/t-cijRNLJbQ7VnsLcafqX33I5nmjDppWNrcJcwqON5UNVrWoz_3S3SsBQAIKSJJKX8F1mGLTnmnKi9S4cf0F-2qTxbV5cDMtEsQmMvVah6ck_qv27iVPp6FsQUX5nyZGzGjlZY4"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation Efficiency </span></b></p>
-<p><b>Gel Extraction and Purification-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Ampicillin backbone  extracted from gel. Machery-Nagel protocol used for gel extraction. </p>
-<p>Weight of gel samples:</p>
-<p>A1 -135.8 mg (271.6 uL of binding buffer NTI used)</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><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A1 - 32.3 ng/uL, 260/280- 1.84</p>
+color:black'>Used 1ul of 50pg/ul &nbsp;of DNA </span></p>
-<p>A2 - 33.4 ng/uL, 260/280- 1.59 </p>
-<p><b>Gibson-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4: 287 / 5*10^-5 = 5.74*10^6 cfu/ug</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5: 328 / 5*10^-5 = 6.56*10^6 cfu/ug</span></p>
-<p><b>Transformation of BH003:</b></p>
+<p class=MsoNormal><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>
+color:black'>6: 414 / 5*10^-5 = 8.28*10^6 cfu/ug</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>
+color:black'>7: 254 / 5*10^-5 = 5.08*10^6 cfu/ug</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><b>8/14/15</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Primers</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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></p>
-<p><b>Transformation-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Prepared PCR of products seen on gel in morning (G1, G2, 001b1,
+b2, 004b1, used 1 uL of leftover PCR reaction). Used Q5 High Fidelity
+polymerase, as no Phusion available. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ingredients:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Q5 High-Fidelity 2X Master Mix- 25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DNA -1 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          F Primer -2.5 uL</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>
+color:black'>          R Primer -2.5 uL</span></p>
-<p>Made 8.5 x of 1X PCR Master Mix:</p>
-<p>	HF Buffer: 85 uL</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>
+color:black'>          H2O -19 uL </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'>&nbsp;</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>
+color:black'>Thermocycler Settings: 98C 30s, 98C 10s, 65C for 30s, 72C for 30s,
-<p><b>	</b>1% Agarose gel run for colony PCR. 1 kB Invitrogen plus ladder used. 50 uL of sample loaded. </p>
+C for 2mins, hold at 4C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>30 cycles </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Prepared Colony PCR To confirm inserts of pMC004,5,6,7. Used Taq
+DNA polymerase instead of phusion. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Pick single colony from plate, place in 50uL of dH2O (acts as DNA
+template)</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add following PCR 1X Master Mix for Taq:</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>5 uL 10X buffer</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>1 uL dNTPs</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>1 uL 10 uM primer stock-VF2 and VR</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>1 uL DNA stock</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>0.5 uL Taq</span></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>41.5 uL H2O </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings: 95C 2mins, 95C 15s, 55C 15s,<span
+style='background:yellow'> 68C 45s</span> (30 cycles), 68C 10m, hold 4C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>-&gt; Extend to 1min per kb (look up on product sheet)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/9/15</span></b></p>
-<p><b>8/17</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Sequences Submitted</b></p>
-<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>
-<p>Primers used: </p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>VF2 (1)</p>
+color:black'>Gel Electrophoresis-</span></b></p>
-<p>VR (2)</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>
+color:black'>Ran 1% Agarose gel 120V, 30 mins. Ran both Colony PCR and Q5 PCR.
+uL each sample loaded. Different ladder used (Quick Load Purple 2-Log from
+NEB. Same amount of EtBr (0.75 uL) used. </span></p>
-<p>Diluted one colony from plates with most growth into 50uL of dH2O to serve as DNA template. </p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2 uL DNA):</p>
+font-family:"Garamond",serif'><br>
-<p>	HF Buffer: 18.00</p>
+<img border=0 width=1262 height=902 id="Picture 13"
-<p>	DMSO: 2.70 uL</p>
+src="..//wiki/images/9/99/UChicago2015Notebook_filesimage013.png"
-<p>	dNTPS: 1.80</p>
+alt="https://lh6.googleusercontent.com/8ID1Wu4cCZQwLMrR4l7op7n00o_k_O7phTHC0tsU2gVfJ1M7bWLOpyTMvvGKtN7T6O0P9AAFRd9gPlqgh7bc7Ju2eNSnCx-p5oshQuhK0kr662ldgZoEARPb_2PHBhPCt0Xy-oc"><br>
-<p>	H2O: 55.</p>
+<br>
-<p>	DNAP: 4.25 uL</p>
+<br>
-<p>	Used 4.50 uL of 10X MC003 and MC004 for mix. </p>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/10/15</span></b></p>
-<p>Thermocycler Settings for Colony </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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>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>
-<p><blockquote>Talked with Justin about ordering materials -TOPO kit should come tomorrow. </blockquote></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></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>
+color:black'>Gel repeated, this time using leftover 45uL of sample. 1kB Plus
-<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>
+invitrogen ladder used. </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'><br>
+<img border=0 width=1094 height=1001 id="Picture 14"
+src="..//wiki/images/e/e9/UChicago2015Notebook_filesimage014.png"
+alt="https://lh4.googleusercontent.com/DIjzha_kO7vq-_NlZnE4aQiO-9rI8Ja38iqhsjQvnnfAiNpJ9t1qBoxcP-r28_jiOA9_tk_W3HHrGTDFducvEWnD0wUejHXyfH7VIUQ8_gsQzq850CGEVFKEiOwND6aZm-xL6Q4"><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Innoculation-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson assembly of BH001_b1 and Cam backbone conducted. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<img border=0 width=1330 height=89 id="Picture 15"
+src="..//wiki/images/0/01/UChicago2015Notebook_filesimage015.png"
+alt="https://lh3.googleusercontent.com/Ro40wlzRHwUwwISfK3r4r6RoQEsg9yIQboeqcm1iqJCQx6qSgQSqdzstLBpIWRx43kS_3ETyfw-RNlrAxvFvBgeVPu5Vs-X8EOlc_n6NdLlav5_rhGWSgjOW3V3m5hTqwUWKvaA"></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Set up PCR for biobricks MC002, and MC003 and BH001
+(confirmation). &nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>11X PCR Master Mix</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Master Mix Recipe-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 110 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 11 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 5.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 341 uL</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>
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 16.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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>
+color:black'>44uL of Master mix, 2.5 uL of F primer, 2.5 uL of R Primer and 1
-<p>MC004/7 -all assembled </p>
+uL of template DNA used. </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 style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<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>
+font-family:"Garamond",serif'>&nbsp;</span></p>
-<ul class=3D"small"><li>MC002_b1</li></ul>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
-<li>MC003_b1</li>
+ style='width:6.5in;border-collapse:collapse'>
-<li>MC002_b1 after PCR</li>
+ <tr>
-<li>MC003_b1 after PCR </li></ul>
+  <td valign=top style='border:solid black 1.0pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>DNA
+  Template</span></b></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>Primers</span></b></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>Info</span></b></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>Gel to Run</span></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC002_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC029, MC010</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1815 bps</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Clone out b1 to give right initial sequence for kaibc/GFP/Term
+  inserts</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1% Agarose</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC003_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC029, MC014</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1815 bps</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Clone out b1 to give right initial sequence for kaibc/GFP/Term
+  inserts</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1% Agarose</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC008_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC003, MC028</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1294 bps</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Clone out kaibc promoter/GFP</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1% Agarose</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Biobrick B0015 Terminator</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC027, MC030</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>129 bps</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Clone out terminator</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2% Agarose</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>BH001/Cam &nbsp;Gibson</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC003, MC004</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>765 bps</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>*should not have done</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>? Was to clone out insert, should have transformed as is. </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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1% Agarose</span></p>
+  </td>
+ </tr>
+</table>
-<p><blockquote>Ligation</blockquote></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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>
+color:black'>Thermocycler settings- 98C 2min, 98C 15s, 67C 30s, 72C 1.5 mins,
-<li>Incubate 5 min at room temperature.   </li>
+C 10min, 4 hold. </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 style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</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>
+color:black'>8/11/15</span></b></p>
-<li>Transfer vials to a 42=C2=B0C water bath for 45 seconds. Immediately return the tubes to ice for 2 min. </li>
-<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'>&nbsp;</span></p>
-<p> pTOPO kit) </p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></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><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=240 height=359 id="Picture 17" src="..//wiki/images/a/a8/UChicago2015Notebook_filesimage016.png"
+alt="https://lh4.googleusercontent.com/usfO-NmUO1TQvxoXM3nWF115qobDFPP21ORF7afn0YMDNK45AGLAfejyjeuwAIkz6WbeigWbHlrF40zR0YKnEye-Br6cvgQY4S3rzPZNPPr_ysLLbqL-05XSAI-J3KEYsljmmAs"><img
+border=0 width=575 height=304 id="Picture 16" src="..//wiki/images/f/f4/UChicago2015Notebook_filesimage017.png"
+alt="https://lh4.googleusercontent.com/LLWlJ3IaCAGROIYBCESSzLg07w49CbpqwO-jiHPXXg_0jTytoaCrpadQQqW9zWtMe6M_d8ai66I1cO0xIZB_q0sjpsYZe5GlQXv6wmApYvj0oYX-c7SnkaaDVsajcRbmUOkwWrM"></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 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>
+color:black'>2% gel for Terminator cloning sample and 1% gel for other PCR
-<p>	Set up 2 50uL 1X PCR Reactions:</p>
+samples (see table) run. 120V for 30 min. </span></p>
-<ul class=3D"small"><li>dNTPS: 1uL</li></ul>
-<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>Thermocycler settings for gBlock PCR</p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</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><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Miniprep</span></b></p>
-<p><b>Innoculation </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>BH protocol</span></b></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><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<li>Pipette all of the colony suspension into the culture tube as well. </li>
-<li>Put in antibiotic to act as primary screening for insert -Cam 1uL, Amp 4uL (antibiotics stored in the -20 fridge) </li>
-<li>Place culture tubes into shaker at 37C overnight (~16 hrs) </li></ol>
-<p><b>Gel Electrophoresis </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>PCR</span></b></p>
-<p><b>Image</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><u><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>PCR of Minipreps</span></u></p>
+<p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>(BH protocol)</span></u></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          <u>PCR of MC002/3 blocks</u></span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>Phusion 7 x of 1X mix</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          HF Buffer -70 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DMSO -10.5 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DNTPs -7 uL </span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          H2O -219 uL</span></p>
-<p><b>8/19/15</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          Phusion DNAP -3.5 uL</span></p>
-<p><b>Transformation Results</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use 44 uL of master mix w/ 2.5 of F and R primers and 1 uL of
+template DNA.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
+ style='width:6.5in;border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>DNA Template</span></b></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><b><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>Primers</span></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>MC002_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC029, MC010</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1815 bps product</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>MC003_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC029, MC014</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1815 bps product</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal align=center style='text-align:center'><span
+  style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>MC008_b1</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>MC003, MC028</span></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1294 bps product</span></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings-</span></p>
-<p><b>Sequencing Results</b></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-<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>
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>65°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1 min (30s x 1.8kb -largest product)</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
-<p><b>Miniprep-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>	</b>Miniprep inoculations 115,116,117,118,125,126,127,128.</p>
-<ol><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. <i>(</i><i><b>*Optional Wash step</i></b><b>)</b></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>115: 91.8 ng/uL, 260/280: 1.90</p>
+color:black'>Thermocycler Settings for Mini-Prep PCR-</span></p>
-<p>116: 172.0 ng/uL, 260/280: 1.82</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>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>120 seconds</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>61.6°C </span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>(NEB - DMSO%*0.8)</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>15 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1 min (30s x 1.8kb -largest product)</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>5 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
-<p><b>PCR </b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p><b>	</b>Set up PCR for both miniprep PCR and PCR of MC002_b1 and MC003_b1 to redo ligation and transformation. </p>
+font-family:"Garamond",serif'><br>
-<p>Set up 2 50uL 1X PCR Reactions for MC002_b1 and MC003_b1:</p>
+<br>
-<li>dNTPS: 1uL</li>
+<br>
-<li>H2O: 31 uL</li>
+</span></p>
-<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><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>	HF Buffer: 18.00</p>
+color:black'>8/12/15</span></b></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 (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'>&nbsp;</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>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>
-<p><b>Gel Electrophoresis-</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:.5in'><u><span
-<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>
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>PCR of
+Terminator </span></u></p>
-<p><b>Image- </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><img src=3D"cid:Image_10.png" /></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:.5in'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>2 50 uL
+Samples &nbsp;</span></p>
-<p><b>8/20</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Sequences Submitted</b></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<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>
+font-family:"Garamond",serif;color:black'>          HF Buffer -10 uL</span></p>
-<p>Primers used: </p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>VF2 (1)</p>
+font-family:"Garamond",serif;color:black'>          DMSO -1.5 uL</span></p>
-<p>MC003 (2)</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 style='margin-left:1.0in'><span style='font-size:16.0pt;
-<p>New primers for MC001 arrived. Proceeded with Gibson assesmbly. Incubated mix at 50C for 1 hour.</p>
+font-family:"Garamond",serif;color:black'>          DNTPs -1 uL </span></p>
-<p><b>PCR</b></p>
-<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>
-<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;color:black'>          H2O -31 uL</span></p>
-<p>	DMSO -11.25 uL</p>
-<p>	DNTPs -7.50 uL </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;color:black'>          Phusion DNAP -0.5 uL</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;color:black'>          10 uM of MC027 Primer -2.5
-<p>MC003_b1 : MC029, MC014</p>
+uL </span></p>
-<p>Gibson rxn : 1b1F, 1b2R</p>
-<p>MC001_b1 : 1b1F, 1b1R</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;color:black'>          10 uM MC030 Primer -2.5 uL</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;color:black'>          DNA from plate -1 uL</span></p>
-<p><b>Gel Electrohporesis-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p><b>8/21</b></p>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>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. </span></p>
-<p><b>PCR</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<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>
-<p>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</p>
+<p class=MsoNormal style='margin-left:.5in'><b><span style='font-size:16.0pt;
-<p>o   Phusion 5X HF Buffer =3D 15 uL</p>
+font-family:"Garamond",serif;color:black'>Gel Electrophoresis-</span></b></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>
+<p class=MsoNormal style='margin-left:.5in'><span style='font-size:16.0pt;
-<p><b>	</b>MC001_b1 and MC001_b2 extracted yesterday were purified. Used Machery-Nagel protocol. </p>
+font-family:"Garamond",serif;color:black'>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% &nbsp;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. </span><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=777 height=569 id="Picture 18" src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage018.png"
+alt="https://lh6.googleusercontent.com/n0sSC5e7IJ5_eaLdIFD-KDmnkktofgffhO0PKExoxa1QerOzJS31nohGqtTT2tEAfUQKpcGITuxC7lb237RDasCzdZcfLnggWBRCsCwEgRANahxD-17xyWhetsq29E9BLThTwj0"></span></p>
-<p>Weights-</p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>1b1 -13.4 mg</p>
-<p>1b2 - 133.4 mg</p>
-<p>Nanodrop results-</p>
+<p class=MsoNormal style='margin-left:.5in'><b><span style='font-size:16.0pt;
-<p>1b1 - 13.1 ng/uL</p>
+font-family:"Garamond",serif;color:black'>Images:</span></b></p>
-<p>1b2 -19.2 ng/ul</p>
-<p><b>Gel Electrophoresis</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
-<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>
+<img border=0 width=457 height=608 id="Picture 19"
+src="..//wiki/images/f/fb/UChicago2015Notebook_filesimage019.png"
+alt="https://lh6.googleusercontent.com/1zI77VjoJucuCfWMqO0uHict0g0ZQNuBdT0iVtFodMNBPyukgpOL7A_C8puF0QKAubva1ehDrgHI-tL7j3B5jYWlbHC35WxPGs2pQzUOQ4AHpMYUKgdCJcERPb4lulpGc4bXSKQ"><br>
+<img border=0 width=644 height=360 id="Picture 20"
+src="..//wiki/images/8/88/UChicago2015Notebook_filesimage020.png"
+alt="https://lh6.googleusercontent.com/wkz8b0Sr1HlJlaJRfdEzNcedYGEFsr1y5kUv0ABe4Ei-bnZihEVt9T3xX37jbl7S6dQLCfNr5Rz7_c2KagMCMtfZlPV11t_aJWP75b1SrtfPyI8eFzGfG_CGKc2jRF45FbpHR6Q"></span></p>
-<p><b>Image-</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<p><img src=3D"cid:Image_11.png" /></p>
+font-family:"Garamond",serif'><br>
-<p><img src=3D"cid:Image_12.png" /></p>
+<br>
+<br>
+</span></p>
-<p><b>Gibson- </b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>	</b>Gibson of MC001 repeated. Incubated mix at 50C for 1 hour. </p>
+color:black'>Gel Purification-</span></b></p>
-<p><b>PCR- </b></p>
+<p class=MsoNormal><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>
+color:black'>Extracted correctly sized product fragmnets under blue light:
-<p>	DMAP: 0.5 uL</p>
+T-189 bps, GFP 1249 bps. </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><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Weights of gels:</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;
+color:black'>GFP 1-63.8 mg</span></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>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>
+color:black'>GFP 2-70.64 mg</span></p>
-<p><b>	</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Gel Electrophoresis-</b></p>
+color:black'>T 1-34.6 mg</span></p>
-<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>
-<p><b>Image-</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T 2-110.0 mg</span></p>
-<p><b>8/24</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</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>
+color:black'>Used Machery-Nagel clean up method:</span></p>
-<p>Miniprep inoculations 115,116,117,118,125,126,127,128.</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<li>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all medium. </li>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<li>Resuspend cells by adding 250uL of resuspension buffer R3 with RNase A. Mix up and down until homogenous. </li>
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
-<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>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>Add 200uL NTI Binding buffer / 100 mg gel </span></p>
-<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. <i>(</i><i><b>*Optional Wash step</i></b><b>)</b></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 style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<p>A21: </p>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A22: </p>
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
-<p>A31:</p>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>A32: </p>
+color:black'>Incubate at 50C for 10 mins</span></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:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transfer solution to spin column and collection tube. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Discard flow through. Add 700 uL of wash buffer NT3 to spin
+column.</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
-<p><b>PCR </b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<p>Gradient PCR of only MC001 blocks conducted. </p>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Discard flow through. Add 700 uL of wash buffer NT3 to spin
+column. </span></p>
-<p>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
-<p>o   Phusion 5X HF Buffer =3D 37.5 uL</p>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   dNTPs 10 mM  =3D 3.75  uL</p>
+color:black'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
-<p>o   Phusion DNAP =3D 1.875 uL</p>
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>o   H2O =3D 116.25 uL</p>
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
-<p>o   DMSO =3D 5.625 uL</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:.5in;text-indent:-.25in;vertical-align:
-<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>
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 1 min to dry silica membrane </span></p>
-<p><b>Image</b></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs)
+for 30s</span></p>
-<p><img src=3D"cid:Image_13.png" /></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop (use EB buffer as blank, load 1.5 uL sample)</span></p>
-<p><b>8/25</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>Sequencing Results: Found a sample (127) containing entire MC001 insert! </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Innoculated sample 127 and prepared for induction with L-Rhamnose. </p>
+color:black'>Purity recorded w/Nanodrop:</span></p>
-<p>Re-plated TOPO reactions for MC002/3 as overgrowth of colonies on previous plates. </p>
-<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>
-<p><b>8/26</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T1- 5.1ng/uL 260/280-14.84</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 style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T2-23.1ng/uL 260/280-1.92</span></p>
-<p>Redo TOPO transformations. </p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>GFP 1- 41.6ng/uL 260/280-1.94</span></p>
-<p><b>8/27</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>GFP 2- 33.1ng/uL 260/280-2.03</span></p>
-<p><b>8/28</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>8/31</b></p>
+<p class=MsoNormal><b><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>
+color:black'>Plan for MC002_b1, MC003_b1: </span></b><span style='font-size:
+.0pt;font-family:"Garamond",serif;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 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.
+</span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mini-Prep Results (Nano-Drop)</span></b></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 style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
-<p>E-mail sleep people</p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=624
+ style='width:6.5in;border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>Sample</span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>260/280</span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>260/230</span></p>
+  </td>
+  <td valign=top style='border:solid black 1.0pt;border-left:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>ng/ul</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>41</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.86</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.20</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>55.1</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>42</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.84</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2.06</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>35.4</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>43</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.89</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.10</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>72.9</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>44</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.92</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.83</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>30.7</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>51</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.94</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.87</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>46.8</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>52</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.90</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2.00</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>53.9</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>53</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.90</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.15</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>66.6</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>54</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.93</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.14</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>58.6</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>61</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.97</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.06</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>52.2</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>62</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.97</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.14</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>61.1</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>63</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2.00</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2.24</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>52.5</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>64</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.92</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>2.12</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>57.1</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>71</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.03</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>2.13</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>56.9</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>72</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.88</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.81</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>50.2</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>73</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.93</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>1.83</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black;background:#FFE599'>59.3</span></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid black 1.0pt;border-top:none;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>74</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.92</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>1.92</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;padding:5.25pt 5.25pt 5.25pt 5.25pt'>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:black'>53.3</span></p>
+  </td>
+ </tr>
+</table>
-<p><b>9/1</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
-<p><b>Induction Solutions</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<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>
+color:black'>The highlighted ones are the ones that we choose to sequence.</span></p>
-<p><b>Culture</b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
-<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>
+font-family:"Garamond",serif'><br>
-<p><b>Dilutions of Rhamnose Inducer Samples</b></p>
+<br>
-<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>
+</span></p>
-<p><b>9/2</b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Minimal Media</b></p>
+color:black'>Sequencing-</span></b></p>
-<p>Ordered M9 minimal media 5X salts on Rust Lab tab. Should take till friday to arrive. </p>
-<p><b>Restriction Digest </b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p>Protocol from iGEM.</p>
+color:black'>The concentration of the DNA templates were too low, so we used 10
-<h2>Digest</h2>
+ug of each.</span></p>
-<ul class=3D"small"><li>Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)</li></ul>
-<ul class=3D"small"><li>5 ul NEB Buffer 2 (use CutSmart)</li></ul>
-<li>0.5 ul BSA</li>
-<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>
+color:black'>The primers were diluted to a 4uM solution from a 100x stock.</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>
+color:black'>(1.4 ul of primer + 33.6 ul of water) </span></p>
-<p><b>Bradford Assay </b></p>
-<p><b>Conducted Western blot </b></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'>&nbsp;</span></p>
-<p><b>Set up Rhamnose time course experiment and rhamnose gradient experiment. Both used M9 media and LB media. M9 media arrived. </b></p>
-<p><b>9/7/15</b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
-<p><b>Miniprepped colony PCR for MC003, BH002, BH003. </b></p>
+color:black'>VF2 [1]</span></p>
-<p><b>9/8/15</b></p>
+<p class=MsoNormal><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>
+color:black'>MC003 (F) [2]</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>
+color:black'>MC041 (F) [3]</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>
+color:black'>MC023 (F) [4]</span></p>
-<!-- autogen end -->
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC022 (R) [5]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>VR [6]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[41]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[43]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[53]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[54]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[62]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[63]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[71]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>[73]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=945 height=340 id="Picture 21"
+src="..//wiki/images/4/4b/UChicago2015Notebook_filesimage021.jpg"
+alt="https://lh6.googleusercontent.com/yywO3_6ssAoFt3Ot5zPOiODBmb_5s3XueMAxaDWCXje-NswHlkYVDNdjcJUL0f06nlGmCrdQNWz3DZH6OknRjkQZX2GAjeRTLqxtdbdimMQ0U4Y1dbxEAkOKTHXQafnFhWf0irE"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>^Things sent.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation of BH001:</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>DNA was transformed into competent cells. RbCl2 competent cells
+used. Efficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>15.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Remove cells from freezer, incubate tubes on ice </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>16.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 1 uL DNA (50 pg//uL) into competent cell tube </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>17.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube on ice for 30 mins</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>18.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube 42C water bath for 1 min heat shock</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>19.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube ice 5 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>20.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>21.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube in shaker at 37C/1100 RPM for 1 hour</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>22.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Heat Cam plate in incubator as cold plate reduces
+efficiency,complete while cells shaking</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>23.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Collect pellet, spin 3000 rcf/gs for 3 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>24.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decant 800 uL of supernatant </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>25.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use glass beads (5-6) per section (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>26.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mix pellet, pipetted 200 uL in total</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>27.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Shake with beads and remove </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>28.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate plate overnight 37C</span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/13/15-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=1410 height=493 id="Picture 22"
+src="..//wiki/images/e/eb/UChicago2015Notebook_filesimage022.png"
+alt="https://lh3.googleusercontent.com/5PRzIarnf4BvFtVUcaOaWS7d_qAewOFHKFvJJvYD4k0uhJCmJEIx757jQ9ru2wKIPW02xwaAK5v_T_oUFl67dbZzkFNBHazN1w9bkjpt1hSfn1Qw_wFuolAG096Kxc7F0k0ZQPA"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation Efficiency of BH001-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>amt dna used/plate</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Plates 1 and 2 were discarded, a colony PCR was done on 6 samples
+from Plate 3</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>They were PCRed separately with two different annealing
+temperatures, 61.6 and 66. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson assembly using 5uL of gibson master mix 2x conducted.
+Assembled on ice. Incubated for 1 hour 50C heatblock.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<img border=0 width=1361 height=81 id="Picture 23"
+src="..//wiki/images/2/20/UChicago2015Notebook_filesimage023.png"
+alt="https://lh5.googleusercontent.com/8zTuzPyZXuqC49j0qL97Utg1_Tn4eW4vcWzlUn88_w52ynrlGdEhfdH4ltnNsUx6ChIfyBv7dr7mchO3S7soqYQtHo1AwHb3YXi4ZffN2UxsdrgkJSbi9oK9tVMzr20TsLL3YF0"></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Prepared 2 50uL 1X PCR samples. </span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          HF Buffer -10 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DMSO -1.5 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DNTPs -1 uL </span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          H2O -31 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          Phusion DNAP -0.5 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          F Primer 10 uM MC003 -2.5
+uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          R Primer 10uM MC004 -2.5 uL
+</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DNA (Gibson assembly rxn
+/13) -1uL </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>65°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 secondsc</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1.65 mins (30s x 3.3kb -largest product)</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings for Mini-Prep PCR- Low Temp</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>120 seconds</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>61.6°C </span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>(NEB - DMSO%*0.8)</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>15 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1 min (30s x 1.8kb -largest product)</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>5 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings for Mini-Prep PCR- High Temp</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>120 seconds</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>66°C </span></u></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>15 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1 min (30s x 1.8kb -largest product)</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>5 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>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).</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image-</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+</span><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=837 height=692 id="Picture 24" src="..//wiki/images/f/fc/UChicago2015Notebook_filesimage024.png"
+alt="https://lh4.googleusercontent.com/lRcesA15fcD4p7N0gVbO38n1_qCfUH__rpM3Jlq_Cw8EiV0VMSIYJEWtSSOCaWaNFRKzBFepZ83Oimx-tdts1d1KlrFtgfMg3TDQqVAGctX3ECHvYCVV0lGdoEujvH254yCRZF0"></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif'><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>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Extraction-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH003 biobrick extracted from gel. Machery-Nagel protocol used for
+gel extraction. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Weight of gel: 85.2 mg</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Used Machery-Nagel clean up method:</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>12.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 200uL NTI Binding buffer / 100 mg gel </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>13.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate at 50C for 10 mins</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>14.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transfer solution to spin column and collection tube. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>15.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>16.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Discard flow through. Add 700 uL of wash buffer NT3 to spin
+column.</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>17.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>18.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Discard flow through. Add 700 uL of wash buffer NT3 to spin
+column. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>19.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 30s</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>20.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Spin at 11,000g (RCFs) for 1 min to dry silica membrane </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>21.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs)
+for 30s</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>22.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop (use EB buffer as blank, load 1.5 uL sample)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop purity- 14.4 ng/uL, 260/280- 1.55</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Made 4 1X 50uL samples to PCR ampicillin
+backbone. Used 1 uL of 25ng/uL amp linearized backbone. Primers MC001 and
+MC002. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Master Mix Recipe-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 50 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 5 &nbsp;uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 2.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 155 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 7.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA (products from Gibson 8/4)= 1.0uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC001= 2.5uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC002 =2.55uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings:98C 30s, 25 cycles: 98C 10s, 69C 30s, 72C
+.5m, 72.0C 10 min, 4C hold</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=1361 height=108 id="Picture 25"
+src="..//wiki/images/4/41/UChicago2015Notebook_filesimage025.png"
+alt="https://lh5.googleusercontent.com/xOvXy9DorVztTDX1w6AnFASvTD8PH2Hxa0ggOORWpHMbMA04L100JIENqG2Rvbo92iDW8wUc5VBZyhZCIeJwZa5SJ39jFKIf22tD4olxWyVXL2dTdTNv-AipORA8bQTHMlt3Oi8"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>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. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image- </span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+</span><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=734 height=463 id="Picture 26" src="..//wiki/images/a/a9/UChicago2015Notebook_filesimage026.png"
+alt="https://lh4.googleusercontent.com/-7lj8c8QrXxqZRW7eACtWpuPUxMEmKh238gYNwhIUyD6E4SVnF6Eq5fM_3CvDe-I4ANqe8yK11jmws019qwa9FNVUapLFRZF0LXuz5yN-uY6cmN7kk73d-XZG_FvyoVKKvXe3UU"></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Extraction and Purification-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ampicillin backbone &nbsp;extracted from gel. Machery-Nagel
+protocol used for gel extraction. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Weight of gel samples:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A1 -135.8 mg (271.6 uL of binding buffer NTI used)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A2 -201.4mg (402.8 uL of binding buffer NTI used) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop concentrations of ampicillin backbone samples</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A1 - 32.3 ng/uL, 260/280- 1.84</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A2 - 33.4 ng/uL, 260/280- 1.59 </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=1361 height=81 id="Picture 27" src="..//wiki/images/a/ab/UChicago2015Notebook_filesimage027.png"
+alt="https://lh6.googleusercontent.com/dufpeo0rHYirlXsq8VIsPbQnu8-K0nIHzB72pqqGtEr_H79ENqE4asCGd9FfDE-xVA9PzD89aB0VHN-MB_az-0qyPpR6Nz0VhZIhpn1t_8dhemST0QvAYAyIRvs7lVE7kLw1V4U"></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation of BH003:</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>DNA was transformed into competent cells. RbCl2 competent cells
+used. Efficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>29.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Remove cells from freezer, incubate tubes on ice </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>30.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 1 uL DNA (50 pg//uL) into competent cell tube </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>31.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube on ice for 30 mins</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>32.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube 42C water bath for 1 min heat shock</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>33.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube ice 5 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>34.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rescue cells by pipetting 900 uL LB into tube (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>35.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate tube in shaker at 37C/1100 RPM for 1 hour</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>36.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Heat Cam plate in incubator as cold plate reduces
+efficiency,complete while cells shaking</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>37.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Collect pellet, spin 3000 rcf/gs for 3 mins </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>38.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Decant 800 uL of supernatant </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>39.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Use glass beads (5-6) per section (use sterile flame)</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>40.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Mix pellet, pipetted 200 uL in total</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>41.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Shake with beads and remove </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>42.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate plate overnight 37C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/14/15</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          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></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<img border=0 width=645 height=540 id="Picture 28"
+src="..//wiki/images/9/9d/UChicago2015Notebook_filesimage028.png"
+alt="https://lh6.googleusercontent.com/5JUbotNvkXgOg9G28elqEXo8vw_hgB6DVsmd1B5eBCtc_X_ViBFGAK6zdzKEYGeQetzG8beXSLk5M5Bni2NjK0FqoG52PhI4ZQ_MG2Tx1Y5-UnGtexg2k_zBNkG2owT9R34hbAk"><br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Colony PCR of BH003/MC001-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Diluted one colony from plates with most growth into 50uL of dH2O
+to serve as DNA template. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Made 8.5 x of 1X PCR Master Mix:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          HF Buffer: 85 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DMSO: 12.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          dNTPS: 8.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          H2O: 263.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DNAP: 4.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Used 2.5uL of VF2 and VR each and 1 uL of sample DNA. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings for Colony </span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 minutes </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>66°C </span></u></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>15 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>2.5 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequencing Primers for MC001- MC003,MC031,MC032,MC006,MC033, MC008</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/15/</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>1% Agarose gel run for colony PCR. 1 kB
+Invitrogen plus ladder used. 50 uL of sample loaded. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<img border=0 width=764 height=592 id="Picture 29"
+src="..//wiki/images/4/46/UChicago2015Notebook_filesimage029.png"
+alt="https://lh3.googleusercontent.com/emrPKYXBDaFV7qpMaymBc932dySXKmseC0tK9A81xnk90xR-KgwSBn9pA4Jt4xjwzp5o5dAHfnua_M_vHcM91nLUKxyCs18p3_henMI20P8fCj3vD1T3gWxxBikJIG-NYA07plo"><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/17</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequences Submitted</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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
+primer:water ratio). 14 uL of miniprepped DNA submitted with 10uL of 4uM
+primers. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Primers used: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>VF2 (1)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>VR (2)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC003 (3)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC031 (4)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC032 (5)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC006 (6)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC033 (7)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC008 (8) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Colony PCR</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Colony PCR of MC001 set up again as upon closer examination,
+insert seems too small to be correct. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Diluted one colony from plates with most growth into 50uL of dH2O
+to serve as DNA template. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix
+and 0.2 uL DNA):</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          HF Buffer: 18.00</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DMSO: 2.70 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          dNTPS: 1.80</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          H2O: 55.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DNAP: 4.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Used 4.50 uL of 10X MC003 and MC004 for mix. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings for Colony </span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 minutes </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>66°C </span></u></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>15 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>2.5 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Talked with Justin about ordering materials -TOPO kit should come
+tomorrow. </span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/18</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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 115,116,117,118,128 could not be loaded. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image-</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<img border=0 width=629 height=408 id="Picture 30"
+src="..//wiki/images/1/15/UChicago2015Notebook_filesimage030.png"
+alt="https://lh5.googleusercontent.com/r6zsedRwioKWtxu0tkFeTi7D_42tqiJY0mG8k6qj8E7kMkYtRZW0w5TzpNGh4g2ICpc5CisNFak7tRlvsuaNWZSWdBa8hctZ45hCaNhC9OlK_JQFQPLFPP04e7R6EMBLR3q8o2E"><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequencing Results</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC001 -still waiting for colony PCR/ specific primers to arrive
+for assembly</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC002/3 -still waiting for TOPO kit to come for transformation
+ligation and assembly</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC004/7 -all assembled </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH001 -assembled, sequence verify </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH002 -gblocks not arrived yet</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH003 -assembled, sequence verify</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH004 -gblocks not arrived</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Zero-Blunt TOPT Ligation and Transformation</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>MC002_b1</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>MC003_b1</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>MC002_b1 after PCR</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>MC003_b1 after PCR </span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ligation</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Combine 2uL product, 1 uL provided salt solution from pTOPO kit,
+uL H20, 1 uL pCR II-Blunt-TOPO vector </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Pipette up and down a few times to mix. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate 5 min at room temperature. &nbsp;&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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, </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thaw one vial of Chemically Competent E. coli cells per
+transformation on ice. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 2&#956;l ligation product to the thawed cells. &nbsp;Keep on
+ice for 30 mins. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transfer vials to a 42°C water bath for 45 seconds. Immediately
+return the tubes to ice for 2 min. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 250&#956;l room temperature SOC orLB to each vial.
+&nbsp;&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate for 1 hr at 37°C. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Plate 200&#956;l cells on LB + kanamycin plates. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Incubate overnight at 37°C</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&nbsp;*The vector confers resistance to kanamycin, NOT ampicillin.
+&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>pTOPO kit) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&nbsp;&nbsp;Note: if you do not have a sufficient number of
+tranformants 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;color:black'>&#8208;</span></i><i><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>30 sec) to
+gently pellet your cells just before plating. &nbsp;Remove 100&#956;l of the
+supernatant, resuspend the cells by pipeting up and down.</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Set up 2 50uL 1X PCR Reactions:</span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>dNTPS: 1uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>H2O: 31 uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DNAP: 0.5 uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DNA: 1 uL (gblocks
+     MC002_b1 and MC003_b1)</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DMSO: 1.5uL </span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>HF Buffer: 10uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>F Primer: 2.5uL
+     (MC029)</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>R Primer: 2.5 uL
+     (MC010 for MC002_b1 and MC014 for MC003_b1)</span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings for gBlock PCR</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>66°C </span></u></p>
+  <p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1.65 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Innoculation </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Colonies from MC001 plates were inoculated as
+colony PCR results were ambiguous. Samples: 115,116,117,118,125,126,127,128. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Aliquot enough LB for 2mL/sample into a Falcon tube. Pipette 2mL
+of LB into each culture tube/sample. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Pipette all of the colony suspension into the culture tube as
+well. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Put in antibiotic to act as primary screening for insert -Cam 1uL,
+Amp 4uL (antibiotics stored in the -20 fridge) </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Place culture tubes into shaker at 37C overnight (~16 hrs) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image</span></b></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<img border=0 width=468 height=609 id="Picture 31"
+src="..//wiki/images/4/47/UChicago2015Notebook_filesimage031.png"
+alt="https://lh6.googleusercontent.com/gsdssnTT8wTdkdP-jkjDqH53bh0FIYeCHl3zd9dceMtuoFXS-9xw9sbQjkiysSL3jwAAWkBECqm1X73yezacNKHin-BvvY7_Skq4KIT-tLDWULzTq83bSFnzUBhfV1zma2itdqg"><br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/19/15</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<img border=0 width=961 height=822 id="Picture 32"
+src="..//wiki/images/f/fb/UChicago2015Notebook_filesimage032.png"
+alt="https://lh4.googleusercontent.com/53EDmknNqpgNN_Znj7zqGc_ubMFs6HmH7Jaw3E17fp-VLhATtdq4jkvgPowy2xAbhDzIl4UmAtpkMNxej-RseZJQdifLyzPOInGIYLkWlSip8j6bfLlEJEkoFV-unPToWnq4i9w"></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformation Results</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequencing Results</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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
+KaiC w/KaiB and suffix) resulted.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Miniprep-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Miniprep inoculations
+,116,117,118,125,126,127,128.</span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove
+all medium. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Resuspend cells by adding 250uL of resuspension buffer R3 with
+RNase A. Mix up and down until homogenous. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. <i>(<b>*Optional
+Wash step</b></i><b>)</b></span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 700 uL of wash buffer W9 with ethanol. centrifuge column at
+,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs.
+Discard flow through. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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. </span></p>
+<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;vertical-align:
+baseline'><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Centrifuge column at 12,000 rcfs for 2 mins. Discard column.
+Nanodrop DNA. Store at 4C short term, -20C long term. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop results-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>115: 91.8 ng/uL, 260/280: 1.90</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>116: 172.0 ng/uL, 260/280: 1.82</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>117: 152.0 ng/uL, 260/280: 1.91</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>118: 120.4 ng/uL, 260/280: 1.81</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>125: 187.0 ng/uL, 260/280: 1.88</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>126: 149.2 ng/uL, 260/280: 1.88</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>127: 211.0 ng/uL, 260/280: 1.87</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>128: 222.8 ng/uL, 260/280: 1.89</span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Set up PCR for both miniprep PCR and PCR of
+MC002_b1 and MC003_b1 to redo ligation and transformation. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Set up 2 50uL 1X PCR Reactions for MC002_b1 and MC003_b1:</span></p>
+<ul style='margin-top:0in' type=disc>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>dNTPS: 1uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>H2O: 31 uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DNAP: 0.5 uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DNA: 1 uL (gblocks MC002_b1
+     and MC003_b1)</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>DMSO: 1.5uL </span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>HF Buffer: 10uL</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>F Primer: 2.5uL
+     (MC029)</span></li>
+ <li class=MsoNormal style='color:black;vertical-align:baseline'><span
+     style='font-size:16.0pt;font-family:"Garamond",serif'>R Primer: 2.5 uL
+     (MC010 for MC002_b1 and MC014 for MC003_b1)</span></li>
+</ul>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix
+and 0.2 uL DNA):</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          HF Buffer: 18.00</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DMSO: 2.70 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          dNTPS: 1.80</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          H2O: 55.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DNAP: 4.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Used 4.50 uL of 10X MC003 and MC004 for mix. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples
+and other for 50uL samples)-</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>67°C </span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1:40 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></p>
+<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image- </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=1180 height=838 id="Picture 33"
+src="..//wiki/images/d/d3/UChicago2015Notebook_filesimage033.png"
+alt="https://lh6.googleusercontent.com/RA4hfJiLGXdMws--G_SPC1NtjjUnOEMIMePRoQnqebfDInZavx4jESYy7N2sKuI9OkX5pfQmUqO3CwG6U8Ov2Okpu8fqSNkS8_uUcBuXezahf8jMUUTHxpdV_i_ekVmKz7O-7_s"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/20</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequences Submitted</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Primers used: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>VF2 (1)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC003 (2)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC031 (3)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC032 (4)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC006 (5)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC033 (6)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC008 (7)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>VR (8) &nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson Assembly</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>New primers for MC001 arrived. Proceeded with Gibson assesmbly.
+Incubated mix at 50C for 1 hour.</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b><span style='font-size:16.0pt;font-family:"Garamond",serif'><img
+border=0 width=1361 height=81 id="Picture 34" src="..//wiki/images/4/46/UChicago2015Notebook_filesimage034.png"
+alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>Phusion 7.5 x of 1X mix</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          HF Buffer -75.00 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DMSO -11.25 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          DNTPs -7.50 uL </span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          H2O -232.50 uL</span></p>
+<p class=MsoNormal style='margin-left:1.0in'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:black'>          Phusion DNAP -3.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 44uL of PCR Mix to 2.5 of F and R Primer each and 1 uL of DNA</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>DNA : Primers</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC002_b1 : MC029, MC010</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC003_b1 : MC029, MC014</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson rxn : 1b1F, 1b2R</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC001_b1 : 1b1F, 1b1R</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>MC002_b2: &nbsp;1b2F, 1b2R </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples
+and other for 50uL samples)-</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>66°C </span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1:30 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrohporesis-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/21</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Due to failure of yesterday’s PCR, gradient PCR
+was set up in order to determine optimal anneal temperature. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 15 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 1.5 &nbsp;uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 0.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 46.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 2.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA =1.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC003= 3.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC004 =3.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>10uL Master mix aliquoted into 7 samples.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup> 15 s,
+<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>, 66<sup>o</sup>, 68<sup>o</sup>,
+<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10
+min, 4<sup>o</sup> hold.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>, 62.0<sup>o</sup>,
+.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>, 72.0<sup>o</sup></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup> for 30 cycles</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Purification </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>MC001_b1 and MC001_b2 extracted yesterday were
+purified. Used Machery-Nagel protocol. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Weights-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1b1 -13.4 mg</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1b2 - 133.4 mg</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop results-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1b1 - 13.1 ng/uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1b2 -19.2 ng/ul</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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.
+colonies chosen for inoculation and miniprep: 3,9 and 22. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=790 height=503 id="Picture 35"
+src="..//wiki/images/5/5e/UChicago2015Notebook_filesimage035.png"
+alt="https://lh5.googleusercontent.com/E5f_qehJxo9MhfwKRgs8YHVlaEh966MaCTP4Xq-KzMGrBoRrVE-LcC-a1YttvHeNVq-9wOmklO8kMx6046adLR488kWfBiTcmmOnmTdqtlgv-nQ4_ALIa1xcrAR1lD9RVmpuWm0"></span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=509 height=663 id="Picture 36"
+src="..//wiki/images/d/de/UChicago2015Notebook_filesimage036.png"
+alt="https://lh4.googleusercontent.com/sUvtKnAEk2sK3xSabkXHLEKeeMi2FUX_zR6SwqW8TfR27XVWG-gk72KBOTbFM87FEBjAzef8k1hNyPRYitVYszT-EDvgJhlVi4C2qAWWzk4WdmgZahr0sqhXlqm0Z-YQnCo6gfo"></span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gibson- </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Gibson of MC001 repeated. Incubated mix at 50C
+for 1 hour. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'><br>
+<img border=0 width=1361 height=81 id="Picture 37"
+src="..//wiki/images/4/46/UChicago2015Notebook_filesimage034.png"
+alt="https://lh3.googleusercontent.com/nVtKwwTZMfpzu9qRZrvLMCGnDD4SijUckeBp3qEuxMjSvz5cB8fdghfllRCqUkxwqLpVA9r4deFL7oKLTzoUAzzbTr0x2tmXQ8SjQi3F5HS8tM2zW-f8ICvPXg6QHxXiz3h08lQ"></span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR- </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DMAP: 0.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DMSO: 1.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          dNTPS: 1 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          DNA: 1uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          HF Buffer: 10 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          H2O: 31 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          F Primer: 2.5 uL (10uM)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          R Primer: 2.5 uL (10uM) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR Conducted one at high anneal temperature (68C) one at low
+anneal temperature (64C)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples
+and other for 50uL samples)-</span></p>
+<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
+ style='border-collapse:collapse'>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>STEP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TEMP </span></u></b></p>
+  </td>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-left:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><b><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>TIME </span></u></b></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Initial Denaturation</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>98°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>30 seconds </span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 Cycles</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>98°C</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>68 or 64°C </span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>10 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>30 seconds</span></u></p>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>1:30 mins</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;background:
+  #ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>Final Extension</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>72°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;background:#ECEDE8;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:#ECEDE8'>10 min</span></u></p>
+  </td>
+ </tr>
+ <tr>
+  <td valign=top style='border:solid #B3B3B3 1.0pt;border-top:none;padding:
+.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>4°C</span></u></p>
+  </td>
+  <td valign=top style='border-top:none;border-left:none;border-bottom:solid #B3B3B3 1.0pt;
+  border-right:solid #B3B3B3 1.0pt;padding:6.0pt 5.25pt 6.0pt 5.25pt'>
+  <p class=MsoNormal><u><span style='font-size:16.0pt;font-family:"Garamond",serif;
+  color:#4C4C4C;background:white'>Hold</span></u></p>
+  </td>
+ </tr>
+</table>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis-</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          </span></b><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>1% agarose gel run at 120V for 30 mins. 1kB Plus invitrogen
+ladder used. 50uL of samples loaded. Results very inconclusive. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/24</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Miniprep-</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>          Inoculated TOPO MC002_b1 and MC003_b1 samples
+miniprepped using invitrogen protocol. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Miniprep inoculations 115,116,117,118,125,126,127,128.</span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>10.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove
+all medium. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>11.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Resuspend cells by adding 250uL of resuspension buffer R3 with
+RNase A. Mix up and down until homogenous. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>12.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>13.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>14.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>15.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. <i>(<b>*Optional
+Wash step</b></i><b>)</b></span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>16.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Add 700 uL of wash buffer W9 with ethanol. centrifuge column at
+,000 gs for 1 min. Discard flow through then dry spin for 1 min at 12,000 gs.
+Discard flow through. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>17.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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. </span></p>
+<p class=MsoNormal style='margin-left:0in;text-indent:0in;vertical-align:baseline'><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:black'>18.<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+</span></span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Centrifuge column at 12,000 rcfs for 2 mins. Discard column.
+Nanodrop DNA. Store at 4C short term, -20C long term. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Nanodrop results-</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A21: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A22: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A31:</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>A32: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>B21: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>B22: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>B31: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>B32: </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>None were sufficient enough to send for sequencing. Likely errors
+also due to overgrowth of colonies. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>PCR </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gradient PCR of only MC001 blocks conducted. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion 5X HF Buffer = 37.5 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>dNTPs 10 mM &nbsp;= 3.75 &nbsp;uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>Phusion DNAP = 1.875 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>H<sub>2</sub>O = 116.25 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DMSO = 5.625 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>DNA =3.75 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>F Primer MC003= 9.375 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>o</span><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'> &nbsp;&nbsp;</span><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:black'>R Primer MC004 =9.375 uL</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>10uL Master mix aliquoted into 7 samples.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Thermocycler settings- 98<sup>o</sup> 2 min, 98<sup>o</sup> 15 s,
+<sup>o</sup><sub>, </sub>62<sup>o</sup>, 64<sup>o</sup>, 66<sup>o</sup>, 68<sup>o</sup>,
+<sup>o</sup>, 72<sup>o</sup> 30 s, 72<sup>o</sup> 1 min, 72<sup>o</sup> 10
+min, 4<sup>o</sup> hold.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Actual Anneal temperatures- 60.0<sup>o</sup>, 62.0<sup>o</sup>,
+.3<sup>o</sup>, 66.6<sup>o</sup>, 68.2<sup>o</sup>, 69.7<sup>o</sup>, 72.0<sup>o</sup></span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>T=66.0<sup>o</sup>, G=6.0<sup>o</sup> for 30 cycles</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Gel Electrophoresis</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>1 % agarose gel run for 30 mins at 120V. 25 uL of samples loaded.
+kB plus Invitrogen ladder loaded. Only sufficient product seen for block 2.
+Indicates issues with block 1. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=615 height=447 id="Picture 38"
+src="..//wiki/images/f/fc/UChicago2015Notebook_filesimage037.png"
+alt="https://lh3.googleusercontent.com/vNACBLm6LidZIh48dOsbDGB6YusPmNCCVzU9Ima_K2Ma8PKbDmeuKbwyH2ZEr69jrCBsMfySsW7latn7jNYFWg66N1xdoZvNoYlJz9EYupv2N3ohTwqP2HvJZ-6Di04qAEWVe_0"></span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/25</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Sequencing Results: Found a sample (127) containing entire MC001
+insert! </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Innoculated sample 127 and prepared for induction with L-Rhamnose.
+</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Re-plated TOPO reactions for MC002/3 as overgrowth of colonies on
+previous plates. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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 </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/26</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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]</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Redo TOPO transformations. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/27</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/28</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>8/31</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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 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.
+</span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH- Type up lysing assay and bradford assay -give some specifics
+about western blot (how much each sample, and calculations) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>E-mail sleep people</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/1</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Induction Solutions</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Culture</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Set up two 40 mL cultures of sample 127 in 250 mL flasks. Added
+uL 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
+C incubator at 11:50. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/2</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Dilutions of Rhamnose Inducer Samples</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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(?) </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Minimal Media</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ordered M9 minimal media 5X salts on Rust Lab tab. Should take
+till friday to arrive. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Restriction Digest </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Protocol from iGEM.</span></p>
+<p class=MsoNormal style='margin-bottom:10.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:#282828;background:white'>Digest</span></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;background:white;
+vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>5 ul NEB Buffer 2 (use CutSmart)</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>0.5 ul BSA</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>0.5 ul </span><a
+href="http://www.neb.com/nebecomm/products/productR3101.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#002BB8;background:
+white'>EcoRI-HF</span></a></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>0.5 ul </span><a
+href="http://www.neb.com/nebecomm/products/productR0140.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#002BB8;background:
+white'>PstI</span></a></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>0.5 ul </span><a
+href="http://www.neb.com/nebecomm/products/productR0176.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#002BB8;background:
+white'>DpnI</span></a><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:#282828;background:white'> (Used to digest any template DNA from
+production)</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>18 ul dH20</span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Digest Plasmid Backbone</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)</span></p>
+<p class=MsoNormal style='margin-left:104.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add 4 ul of Enzyme Master Mix</span></p>
+<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:2.0pt;
+margin-left:104.25pt;text-indent:-.25in;background:white;vertical-align:baseline'><span
+style='font-size:10.0pt;font-family:Wingdings;color:#282828'>§<span
+style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Digest 37C/30 min, heat kill 80C/20 min</span></p>
+<p class=MsoNormal style='margin-bottom:10.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif;color:#282828;background:white'>Ligation</span></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;background:white;
+vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add 2ul of digested plasmid backbone (25 ng)</span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add 1 ul </span><a
+href="http://www.neb.com/nebecomm/products/productm0202.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#002BB8;background:
+white'>T4 DNA ligase buffer</span></a><span style='font-size:16.0pt;font-family:
+"Garamond",serif;color:#282828;background:white'>. <b>Note:</b> Do not use
+quick ligase</span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add 0.5 ul </span><a
+href="http://www.neb.com/nebecomm/products/productm0202.asp"><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#002BB8;background:
+white'>T4 DNA ligase</span></a></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Add water to 10 ul</span></p>
+<p class=MsoNormal style='margin-left:53.25pt;text-indent:-.25in;background:
+white;vertical-align:baseline'><span style='font-size:10.0pt;font-family:Wingdings;
+color:#282828'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Ligate 16C/30 min, heat kill 80C/20 min</span></p>
+<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:1.0pt;
+margin-left:53.25pt;text-indent:-.25in;background:white;vertical-align:baseline'><span
+style='font-size:10.0pt;font-family:Wingdings;color:#282828'>§<span
+style='font:7.0pt "Times New Roman"'>&nbsp; </span></span><span
+style='font-size:16.0pt;font-family:"Garamond",serif;color:#282828;background:
+white'>Transform with 1-2 ul of product</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Restreaked TOPO plate</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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 miniprepping, these colonies were streaked onto new Cam plates.
+&nbsp;&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transformed new TOPO reactions for MC02/3 and BH002/3</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>New TOPO reactions were conducted in parallel with the streaking. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Set up Gibson of BH002/3</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>BH002 and BH003 were gibsonsed with their respective Amp backbones
+and Cam backbones. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/3 </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Lysed cells after induction </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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
+s and incuabted on ice for 1 min (repeated 5 times). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford Assay </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford assay conducted to quanity amount of protein added. 1 mL
+x 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). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Conducted Western blot </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>SDS-Page gel</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transfer</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Blocking and Staining</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ran colony PCR of TOPO reactions</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Only MC003 TOPO worked. BH002/3 Gibsons worked, no TOPO results
+&nbsp;for MC002, BH002, BH003. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Inoculated negative control</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ecoli with vector (Cam backbone) innoculated at 37C. 2 mL
+innoculation generated. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Image</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=917 height=279 id="Picture 39"
+src="..//wiki/images/e/ed/UChicago2015Notebook_filesimage038.png"
+alt="https://lh4.googleusercontent.com/qi6z0XeDqFcoAaomNv3uJYjNWeOkAjg9dWljPlg474qH2PgOCbVWStI-wwSYG4AxtZKZRKqFVHE-YPjUVuc6RKDlC6wNrP-9EizaWbl7cTfvD8M3k3ggW97-kzMdkMMMVg=s1600"></span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>KaiB and KaiA gave no clear results. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/4/15</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Set up Rhamnose time course experiment and rhamnose gradient </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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
+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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Rhamnose gradient assay also conducted, using smaller gradient
+with 10 fold dilution. Induction conducted for 10 hours. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/7/15</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/8/15</span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford Assay </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford assay conducted to quanity amount of protein added. 1 mL
+x 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). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Conducted Western blot </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>SDS-Page gel</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/9</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Blocking and Staining</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=1506 height=910 id="Picture 40"
+src="..//wiki/images/f/f7/UChicago2015Notebook_filesimage039.png"
+alt="https://lh6.googleusercontent.com/SPybomQoqbf7c2PeVmAFpbrVANHCm5VAEpe6J2nuDqeXm2T5Kqb7YxjUIcdDBmv3O0XWM3OPUHn9kOsBKej490C6Qzh3JiNuHbM_PP_34-EwtQ-q7bbUw4-4wBS-q__gxw=s1600"></span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/10</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'><img border=0 width=1073 height=271 id="Picture 41"
+src="..//wiki/images/3/36/UChicago2015Notebook_filesimage040.png"
+alt="https://lh4.googleusercontent.com/xsJGDEr3-BGG075g4bYfif1LUKzwP9hUAn2Sg0E1ZY5Br9Gg5ypX0FwcqgmTkhKsaLjU-x3qLo1cQ7DPU7eP3PM3S-Eer2pKTI-AKrcDNy-yOVWIWyz8hqLYqSf0ovDkrg=s1600"></span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/11 </span></b></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford Assay </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Bradford assay conducted to quanity amount of protein added. 1 mL
+x 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). </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Conducted Western blot </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>SDS-Page gel</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Transfer</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ran another PCR of MC002_b1, MC003_b1</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Ran PCR of gel extracted blocks MC002_b1 and MC003_b1</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/12 </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><i><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Blocking and Staining</span></i></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>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. </span></p>
+<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:16.0pt;
+font-family:"Garamond",serif'><br>
+<br>
+</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/13 </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>No primary antibody available at time, continued to block. </span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/14 </span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+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></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>9/15</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Induction</span></b></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Induction repeated. Cells pelleted, washed with 1 mL water,
+pelleted then reintroduced into Lb or M9 media.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>&nbsp;</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif;
+color:black'>Although this as much data as we have as of a few days before the
+Wiki freeze, we will definitely have important updates to come we hope to
+present during the Jamboree.</span></p>
+<p class=MsoNormal><span style='font-size:16.0pt;font-family:"Garamond",serif'>&nbsp;</span></p>
+</div>
+</body>
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