Difference between revisions of "Team:UChicago/Notebook"

Revision as of 03:32, 14 September 2015

Notebook

Document the dates you worked on your project.

What should this page have?

Chronological notes of what your team is doing.
Brief descriptions of daily important events.
Pictures of your progress.
Mention who participated in what task.

Inspiration

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GeneHackers Summer 2015 Journal

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 direc= tion 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 =E2= =80=9CGenehackers 2015 Inventory=E2=80=9D

Downloaded SnapGene Viewer to work on plasmid constructs.

6/16/15

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

Read-out activator (KaiCEE-RFP, RpaA, SasA)
Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)
Test gene (kaibc promoter, GFP)
Fusion SasA
Fusion KaiC-P

Last two based heavily on paper (Chen, 2015)

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

To Discuss: Is it worth having negative feedback regulator of RpaA/ o= utput molecule?

6/17/15

Decided to use CikA as negative regulator/inhibitor instead of Lab A. Ci= kA better characterized. Reviewed (Gutu. O=E2=80=99Shea, 2013).

6/18/15

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

To Discuss: Specific RBS and promoter strengths on different genes. <= /b>

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=3D 100ng/uL

Transformed plate- PJ006 MC 6/18/15

Steps

Remove cells from freezer, incubate on ice

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

Incubate tube on ice for 30 mins

Incubate tube 42C water bath for 1 min heat shock

Incubate tube ice 5 mins

Rescue cells by pipetting 900 uL LB into tube (use sterile flame)
<= /ol>
*Used LB from MJR Lab, will have to make LB tomorrow

Incubate tube in shaker 37C for 1 hour

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

Collect pellet, spin 3000 rcf/gs for 3 mins

Decant 800 uL of supernatant

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

Mix pellet, pipetted 200 uL in total, 180 to 0.90 section, 20 to 0.10 s= ection

Shake with beads and remove

Incubate plate overnight 37C

6/19/15

Checked on Transformed Plates

Good growth on both sections.

Transformation efficiency

0.10 Section

=3D (293 cfus) / ( ((1 uL x 100 ng/uL)/1000 uL soln)(20/200 uL pla= ted))

=3D 293 cfus/0.01 ng DNA plated =3D 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=3D 6 C, Chloramphenicol storage temperature=3D 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 Invent= ory)

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.col= i procedure -protocol under master list titled =E2=80=9CCompetent E.Coli 6/= 21/15=E2=80=9D

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:

=E2=86=92 Discussed project direction and construct design. Need to add = terminator after RpaA. Need to decide whether or not fusion protein constru= ct worth it. Given that RpaA might have some basal phosphorylation level, i= nduced 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 b= io-brick. Final biobrick would possibly only have CikA, SasA. Overall conse= nsus is that CikA worth exploring. Need to develop primers ASAP.

=E2=86=92 Need to develop more work on biosynthesis pathway and decide w= hich molecule want to consider as well as what is focus of experiment. Want= KaiABC as submitted biobrick so perhaps focusing on biosynthesis pathway i= s a bit ambitious. Need to consider perhaps alternative, simpler molecule. =

=E2=86=92 Will likely assemble using Gibson, order this free kit from RP= I.

Contact Danny for competent cells and Justin for Gibson primers

6/24/15

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

6/25/15

Finished designing primers, will check with Kevin tomorrow. Inoculated t= wo 5mL LB broths with 1-3 colonies each. NEB Gibson Assembly Kit with comp= etent cells arrived!

6/26/15

Carried out competent cell procedure.

http://www.unc.edu/depts/marz= luff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20B= L21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf

https://drive.google.com/open?id=3D0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxP= U01uclJCVmNV

Used both Rust Lab and RbCl2 as a comparison.

Week 3:

Goals- Test efficiency of competent cells, start as much cloning as p= ossible

6/29/15

Conducted Transformation of CaCl2 and RbCl2.

Remove cells from freezer, incubate tubes on ice

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

Incubate tube on ice for 30 mins

Incubate tube 42C water bath for 1 min heat shock

Incubate tube ice 5 mins

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

Incubate tube in shaker 37C for 1 hour

Heat Cam plate in incubator as cold plate reduces efficiency,complete w= hile cells shaking

Collect pellet, spin 3000 rcf/gs for 3 mins

Decant 800 uL of supernatant

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

Mix pellet, pipetted 200 uL in total

Shake with beads and remove

Incubate plate overnight 37C

Spec on LB negative control culture overnight =3D-0.019 A (no growth at = all)

=3D (52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/20= 0 uL plated))

=3D 52 cfus/(4.5 x 10^-5) ng DNA plated =3D1.15 x 10^6 transf= ormants/ng (Rust)

=3D52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 = uL plated))

=3D 18 cfus/(4.5 x 10^-5) ng DNA plated =3D4.00 x 10^5 transf= ormants/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. A= lso 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 Osc= illation system were designed as well.

Week 4:

Goals- Order final gBlocks and primers. Standardize and develop speci= fic, in depth protocols. Practice Western Blots, start writing project repo= rt.

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. Prime= rs were designed.

7/22/15

Primers ordered. Reached out to grad advisers for Western Blotting techn= iques. 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 c= learly 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 plas= mids and background info for wiki and presentation, order materials, gblock= assembly

7/28

Western blot procedures expanded. See Aaron=E2=80=99s email about compat= ible backbones. This week discussed assays -will need to western blot for K= aiA before investigating oscillatory system in order to characterize input = L-Rhamnose to output Kai A production. Dilutions will occur on log scale fi= rst for L-Rhamnose.

7/29

Finished specific protocols -need to ask White lab for sonicator?. Looke= d up compatibility of backbones. pSB1,3,4 have pMB1 (copy number 100-300/ce= ll), p15A (low-medium 10-12 copy), pSC101 (~5 copies/cell). Will need to co= nstruct 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 min= imize 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 st= andardized amount 50 ng of largest block in each assembly. Assembled on ice= . Incubated on 50oC heatblock for 1 hour.

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 o= f Master Mix with 1 uL from Gibson Assembly Mix.

Recipe Phusion Master Mix:

o Phusion 5X GC Buffer =3D 10uL x 5.5 =3D 55 uL

o dNTPs 10 mM =3D 1 x 5.5 =3D5.5 uL

o F Primer MC003 10 uM =3D 2.5 x 5.5 =3D13.75 uL

o R Primer MC004 10 uM =3D 2.5 x 5.5=3D 13.75 uL

o Phusion DNAP =3D 0.5 x 5.5 =3D2.75 uL

o H2O =3D 32.5 x 178.75 uL

o DMSO =3D 1.5 x 5.5 =3D8.25 uL

*Should have added only 170.5 uL (31 x 5.5) =E2=80=93Mix slightly more = dilute

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

Week 6:

Goals- gblock assembly and Transformation

8/3/15

Decided on backbones:

Oscillator MC001 =E2=80=93 Cmr (standard igem backbo= ne for submitted biobrick)

Readout SasA MC002 =E2=80=93Amp (theoretically want to use with MC001 if= successful) =C3=A0 need to add GFP + kai bc

Readout SasA/CikA MC003 =E2=80=93Amp (=E2=80=9C =E2=80=9C)= =C3=A0 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 TA= E 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 1= 5-20 mins.

Gel Electrophoresis

Loaded 10uL of 6X loading dye into 50uL samples. Loaded 10 uL of 1 kB Pl= us 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 issu= e with evaporation in thermocycler. Gel too big for tray. Yields of product= s exist, however seems low. Issue with MC001 =E2=80=93no clear product visi= ble. PCR should be redone.

Image:

Gel Extraction

Gel Weights:

MC001 =E2=80=93N/A

MC004 -0.1536 g

MC005 -0.2048 g

MC006 -0.0965 g

MC007 -0.3962 g

*1mg =3D 1uL

Added 3x uL QG buffer to volume of gel and 1X uL of isopropanol to volum= e 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 =3D 150 uL

o dNTPs 10 mM =3D 15 uL

o Phusion DNAP =3D 7.5 uL

o H2O =3D 465 uL

o DMSO =3D 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- 98o 2 min, 98o 15 s, 69o 30 s, 72o 1 m= in, 72o 10 min, 4o 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 backbone= s. Products from MC004,5,6,7 and Cmr Linearized backbones extracted using g= el punches (borrowed from Rust Lab, need to order more to return). MC001 st= ill not very good yield. Next step to purify MC004-7 and linearized backbon= es, 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, 3.4= 305 uL H2O, heatblock for 1 hour 50oC.

PCR

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

o Phusion 5X HC Buffer =3D 14 uL

o dNTPs 10 mM =3D 1.4 uL

o Phusion DNAP =3D 0.7 uL

o H2O =3D 43.4 uL

o DMSO =3D 2.1 uL

o DNA =3D1.4 uL

o F Primer MC003=3D 3.5 uL

o R Primer MC004 =3D3.5 uL

10uL Master mix aliquoted into 7 samples.

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.

Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72= .0o

T=3D66.0o, G=3D6.0o for 30 cycles

8/5/15

Gel Electrophoresis

Made 90 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye). Ru= n for 120V, 30 mins. EtBr cloud on gel seen, only ladder shows visible band= s. 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=E2=80=99 -

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 =3D 15 uL

o dNTPs 10 mM =3D 15 uL

o Phusion DNAP =3D .75 uL

o H2O =3D 46.5 uL

o DMSO =3D 2.25 uL

o DNA (products from Gibson 8/4)=3D 1.5uL

o F Primer MC003=3D 3.75uL

o R Primer MC004 =3D3.75uL

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 =3D 10 uL

o dNTPs 10 mM =3D 1 uL

o Phusion DNAP =3D .5 uL

o H2O=3D 31 uL

o DMSO =3D 1.5 uL

o DNA (pMC001_b1)=3D 1 uL

o F Primer MC005=3D 2.5uL

o R Primer MC006 =3D2.5uL

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, 60o, 62o, 64o, 66o, 68o, 70o= , 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.

Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72= .0o

T=3D66.0o, G=3D6.0o for 30 cycles

8/6/15

Gel Electrophoresis

Made 100 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye) an= d 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 sho= ws faint products under 68,70, and 72o. Could mean issue with primers. Stra= ngely, no product of right gBlock size seen. Again could be primers.

Image:

Gibson Assembly:

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

Transformation of Assembled products:

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

Remove cells from freezer, incubate tubes on ice

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

Incubate tube on ice for 30 mins

Incubate tube 42C water bath for 1 min heat shock

Incubate tube ice 5 mins

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

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

Heat Cam plate in incubator as cold plate reduces efficiency,complete w= hile cells shaking

Collect pellet, spin 3000 rcf/gs for 3 mins

Decant 800 uL of supernatant

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

Mix pellet, pipetted 200 uL in total

Shake with beads and remove

Incubate plate overnight 37C

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

PCR

PCR of 8/3 Gibson PCR, 8/4 Gibson PCR conducted for further amplificatio= n. 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 e= ach PCR (5 samples in total).

Added Ingredients to individual Samples

o Phusion 5X HC Buffer =3D 10 uL

o dNTPs 10 mM =3D 1 uL

o Phusion DNAP =3D 0.5 uL

o H2O =3D 31.0 uL

o DMSO =3D 1.5 uL

o DNA =3D1.0 uL

o F Primer =3D 2.5 uL

o R Primer =3D2.5 uL

10uL Master mix aliquoted into 7 samples.

Thermocycler settings- 98o 2 min, 98o 15 s, 70o, 30 s, 72o 1 min, 72o 10= min, 4o 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 unde= r blue light. Under UV light, products seemed more specific. Still not as s= trong as in previous gels. Perhaps need to troubleshoot PCR better.

Image:

Transformation Results

Used iPhone application =E2=80=9CColonyCount=E2=80=9D to assist in count= ing 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 =3D 5.74*10^6 cfu/ug

5: 328 / 5*10^-5 =3D 6.56*10^6 cfu/ug

6: 414 / 5*10^-5 =3D 8.28*10^6 cfu/ug

7: 254 / 5*10^-5 =3D 5.08*10^6 cfu/ug

Primers

Designed Sequencing primers as well as new primers for pMC001. Primers m= ade specifically for oscillator plasmid -overhangs incorporated to make pri= mers longer and more specific.

PCR

Prepared PCR of products seen on gel in morning (G1, G2, 001b1, 001b2, 0= 04b1, 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 f= or 2mins, hold at 4C

30 cycles

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

Pick single colony from plate, place in 50uL of dH2O (acts as DNA t= emplate)

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 ea= ch 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 invitrog= en ladder used.

Innoculation-

Due to failure of Colony PCR, colonies were inoculated and incubated. Wi= ll conduct direct miniprep on 8/11 and sequence to sequence. This should he= lp 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 =3D 110 uL

o dNTPs 10 mM =3D 11 uL

o Phusion DNAP =3D 5.5 uL

o H2O =3D 341 uL

o DMSO =3D 16.5 uL

44uL of Master mix, 2.5 uL of F primer, 2.5 uL of R Primer and 1 uL of t= emplate 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% Agar= ose
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 promote= r/GFP
1% Agarose
Biobrick B0015 Terminator
MC027, MC030
129 bps
Clone out terminator
2% Agarose
BH001/Cam G= ibson
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 10m= in, 4 hold.

8/11/15

Gel Electrophoresis-

2% gel for Terminator cloning sample and 1% gel for other PCR samples (s= ee 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 D= NA.

DNA Template
Primers
MC002_b1
MC029, MC0= 10
1815 bps product
MC003_b1
MC029, MC014
1815 bps = product
MC008_b1
MC003, MC028
1294 bps product

Thermocycler Settings-

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
<= p>65=C2=B0C
72=C2=B0C
10 seconds
30 seconds
1 min (= 30s x 1.8kb -largest product)
Final Extension
72=C2=B0C
1= 0 min
Hold
4=C2=B0C
Hold

Thermocycler Settings for Mini-Prep PCR-

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
120 seconds
30 Cycles
98=C2=B0C
=
61.6=C2=B0C
(NEB - DMSO%*0.8)
72=C2=B0C
15 seconds
30 seconds
1 min (30s x 1.8kb -largest product)
Final Exten= sion
72=C2=B0C
5 min
Hold
4=C2=B0C
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, 6= 9C 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 w= ell as MC002/3 clone parts PCR. 25 uL of sample loaded for Terminator 2% g= el. 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 4= 1,43,51,52,53,54,61,62,63,64,74 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:

Add 200uL NTI Binding buffer / 100 mg gel

Incubate at 50C for 10 mins

Transfer solution to spin column and collection tube.

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

Discard flow through. Add 700 uL of wash buffer NT3 to spin column.
Spin at 11,000g (RCFs) for 30s

Discard flow through. Add 700 uL of wash buffer NT3 to spin column.
Spin at 11,000g (RCFs) for 30s

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

Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 3= 0s

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 probabl= y due to repeating regions of DNA in blocks 1 of MC002 and MC003. As IDT ex= pressed, there are a lot of low mass products that are more efficiency ampl= ified by primers. Therefore, as Jennifer Moran mentioned, the best course o= f action would be to insert these two gblocks into a Zero Blunt TOPO PCR Ve= ctor and transforming into competent cells to amplify our gblocks. After pr= oducing colonies, we can PCR and then screen for colonies with correct prod= uct size. We will then miniprep and send these blocks in for sequencing. Th= is will take more time, but will ensure the purity of the insert. After con= firming the correct sequence, the DNA from the miniprep/gel extraction? can= be used to gibson the GFP/kaibc, the terminator, and the first blocks toge= ther.

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
5= 1
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<= /p>
58.6
61
1.97
2.06
52.2
62
1.97
2.14
61.1
63
2.00
2.24
52.5
64
<= p>1.92
2.12
57.1
71
2.03
2.13
56.9
<= p>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. Ef= ficiency of cells: 4.00 x 10^5 transformants/ng (RbCl2)

Remove cells from freezer, incubate tubes on ice

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

Incubate tube on ice for 30 mins

Incubate tube 42C water bath for 1 min heat shock

Incubate tube ice 5 mins

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

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

Heat Cam plate in incubator as cold plate reduces efficiency,complete w= hile cells shaking

Collect pellet, spin 3000 rcf/gs for 3 mins

Decant 800 uL of supernatant

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

Mix pellet, pipetted 200 uL in total

Shake with beads and remove

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 P= late 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 o= n 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 Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
=
65=C2=B0C
72=C2=B0C
10 seconds
30 secondsc
1.65 = mins (30s x 3.3kb -largest product)
Final Extension
72=C2=B0C
10 min
Hold
4=C2=B0C
Hold

Thermocycler Settings for Mini-Prep PCR- Low Temp

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
120 seconds
30 Cycles
98=C2=B0C
=
61.6=C2=B0C
(NEB - DMSO%*0.8)
72=C2=B0C
15 seconds
30 seconds
1 min (30s x 1.8kb -largest product)
Final Exten= sion
72=C2=B0C
5 min
Hold
4=C2=B0C
Hold

Thermocycler Settings for Mini-Prep PCR- High Temp

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
120 seconds
30 Cycles
98=C2=B0C
=
66=C2=B0C
72=C2=B0C
15 seconds
30 seconds
1 min= (30s x 1.8kb -largest product)
Final Extension
72=C2=B0C
5 min
Hold
4=C2=B0C
Hold

Gel Electrophoresis-

100 mL of 1% agarose gel run 120V, 30 mins. Samples loaded inclu= de 45 uL of BH003 biobrick and 20 uL of colony PCRs. 5 uL 1 kB plus Invitro= gen 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 e= xtraction.

Weight of gel: 85.2 mg

Used Machery-Nagel clean up method:

Add 200uL NTI Binding buffer / 100 mg gel

Incubate at 50C for 10 mins

Transfer solution to spin column and collection tube.

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

Discard flow through. Add 700 uL of wash buffer NT3 to spin column.
Spin at 11,000g (RCFs) for 30s

Discard flow through. Add 700 uL of wash buffer NT3 to spin column.
Spin at 11,000g (RCFs) for 30s

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

Elute DNA with 15 uL of NE elution buffer. Spin at 11,000g (RCFs) for 3= 0s

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 =3D 50 uL

o dNTPs 10 mM =3D 5 uL

o Phusion DNAP =3D 2.5 uL

o H2O =3D 155 uL

o DMSO =3D 7.5 uL

o DNA (products from Gibson 8/4)=3D 1.0uL

o F Primer MC001=3D 2.5uL

o R Primer MC002 =3D2.55uL

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 t= o try assemble pMC001 and transform directly. Reaction incubated on heatblo= ck 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 us= ed. 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 5= uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hou= r 50C heatblock.

Transformation of BH003:

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

Remove cells from freezer, incubate tubes on ice

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

Incubate tube on ice for 30 mins

Incubate tube 42C water bath for 1 min heat shock

Incubate tube ice 5 mins

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

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

Heat Cam plate in incubator as cold plate reduces efficiency,complete w= hile cells shaking

Collect pellet, spin 3000 rcf/gs for 3 mins

Decant 800 uL of supernatant

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

Mix pellet, pipetted 200 uL in total

Shake with beads and remove

Incubate plate overnight 37C

8/14/15

Transformation-

MC001 and BH003 gave good results from transformation. Colonies were sm= all, but evenly distributed. Colonies could be small because plates incubat= ed late last night.

Colony PCR of BH003/MC001-

Diluted one colony from plates with most growth into 50uL of dH2O to ser= ve 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 Denatur= ation
98=C2=B0C
10 minutes
30 Cycles
98=C2=B0C
=
66=C2=B0C
72=C2=B0C
15 seconds
30 seconds
2.5 m= ins
Final Extension
72=C2=B0C
10 min
Hold
4= =C2=B0C
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 u= sed. 50 uL of sample loaded.

8/17

Sequences Submitted

MC001 note submitted on weekend for sequencing. MC001 submitted in morni= ng 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 seem= s too small to be correct.

Diluted one colony from plates with most growth into 50uL of dH2O to ser= ve 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 Denatur= ation
98=C2=B0C
10 minutes
30 Cycles
98=C2=B0C
=
66=C2=B0C
72=C2=B0C
15 seconds
30 seconds
2.5 m= ins
Final Extension
72=C2=B0C
10 min
Hold
4= =C2=B0C
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 res= ults from colony PCR, indicating something wrong during PCR. Likely issue w= ith 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 eva= poration in thermocycler, which is why samples 115,116,117,118,128 could no= t be loaded.

Image-

Sequencing Results

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

MC001 -still waiting for colony PCR/ specific primers to arrive for asse= mbly

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 al= low the bacteria to amplify blocks 1 of MC002 and MC003.

MC002_b1

MC003_b1

MC002_b1 after PCR

MC003_b1 after PCR

Ligation

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

Pipette up and down a few times to mix.

Incubate 5 min at room temperature.

No overnight incubation needed! But you can leave it overnight at 4=C2= =B0C if you cannot proceed with the transformation right away,

Transformation

Thaw one vial of Chemically Competent E. coli cells per transformat= ion on ice.

Add 2=CE=BCl ligation product to the thawed cells. Keep on ice for 30 = mins.

Transfer vials to a 42=C2=B0C water bath for 45 seconds. Immediately re= turn the tubes to ice for 2 min.

Add 250=CE=BCl room temperature SOC orLB to each vial.

Incubate for 1 hr at 37=C2=B0C.

Plate 200=CE=BCl cells on LB + kanamycin plates.

Incubate overnight at 37=C2=B0C

*The vector confers resistance to kanamycin, NOT ampicillin.

pTOPO kit)

Note: if you do not have a sufficient number of tranformants in 20= 0=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 su= pernatant, 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 Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
=
66=C2=B0C
72=C2=B0C
10 seconds
30 seconds
1.65 = mins
Final Extension
72=C2=B0C
10 min
Hold
4= =C2=B0C
Hold

Innoculation

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

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

Pipette all of the colony suspension into the culture tube as well.
Put in antibiotic to act as primary screening for insert -Cam 1uL, Amp = 4uL (antibiotics stored in the -20 fridge)

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 produ= ct 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 i= f cells are very efficient as cells could also be very conducive to transfo= rmation.

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.

Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove a= ll medium.

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

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

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

Load supernatant (750 uL) into Spin column (machery nagel used) and col= lection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow thro= ugh and place column back in wash tube.

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)

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. Di= scard flow through.

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

Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop D= NA. 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_b= 1 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 ot= her for 50uL samples)-

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
=
67=C2=B0C
72=C2=B0C
10 seconds
30 seconds
1:40 = mins
Final Extension
72=C2=B0C
10 min
Hold
4= =C2=B0C
Hold

Gel Electrophoresis-

1% agarose gel run at 120V for 30 mins. Loaded 10uL and 45 uL for minipr= eps and MC002/3 gel extractions respectively. Loaded 5uL of 1kB Plus Invitr= ogen 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 s= ubmitted 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. Incubate= d 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 u= sed 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 ot= her for 50uL samples)-

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
=
66=C2=B0C
72=C2=B0C
10 seconds
30 seconds
1:30 = mins
Final Extension
72=C2=B0C
10 min
Hold
4= =C2=B0C
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 extracte= d separately.

8/21

PCR

Due to failure of yesterday=E2=80=99s 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 =3D 15 uL

o dNTPs 10 mM =3D 1.5 uL

o Phusion DNAP =3D 0.75 uL

o H2O =3D 46.5 uL

o DMSO =3D 2.25 uL

o DNA =3D1.5 uL

o F Primer MC003=3D 3.75 uL

o R Primer MC004 =3D3.75 uL

10uL Master mix aliquoted into 7 samples.

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.

Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72= .0o

T=3D66.0o, G=3D6.0o for 30 cycles

Gel Purification

MC001_b1 and MC001_b2 extracted yesterday were purified. Used Ma= chery-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 Invit= rogen ladder used. 10uL of sample loaded. Expected product size ~3000 bps. = No expected product size appeared on gel. Perhaps indicates issues with Gib= son assembly. 24 inoculated colony PCRs also run on separate gel. 3 coloni= es 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 (MC00= 1_b2_F_new, MC001_b2_R_new), MC001 Gibson (MC001_b1_F_new, MC001_b2_R_new)<= /p>
PCR Conducted one at high anneal temperature (68C) one at low anneal tem= perature (64C)

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

STEP
TEMP
TIME
Initial Denatur= ation
98=C2=B0C
30 seconds
30 Cycles
98=C2=B0C
=
68 or 64=C2=B0C
72=C2=B0C
10 seconds
30 seconds
1:30 mins
Final Extension
72=C2=B0C
10 min
Hold
4=C2=B0C
Hold

Gel Electrophoresis-

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

Image-

8/24

Miniprep-

Inoculated TOPO MC002_b1 and MC003_b1 samples miniprepped using invitro= gen protocol.

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

Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all m= edium.

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

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

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

Load supernatant (750 uL) into Spin column (machery nagel used) and col= lection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow thro= ugh and place column back in wash tube.

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)

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. Di= scard flow through.

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

Centrifuge column at 12,000 rcfs for 2 mins. Discard column. Nanodrop D= NA. 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 d= ue 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 =3D 37.5 uL

o dNTPs 10 mM =3D 3.75 uL

o Phusion DNAP =3D 1.875 uL

o H2O =3D 116.25 uL

o DMSO =3D 5.625 uL

o DNA =3D3.75 uL

o F Primer MC003=3D 9.375 uL

o R Primer MC004 =3D9.375 uL

10uL Master mix aliquoted into 7 samples.

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.

Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72= .0o

T=3D66.0o, G=3D6.0o for 30 cycles

Gel Electrophoresis

1 % agarose gel run for 30 mins at 120V. 25 uL of samples loaded. 1kB pl= us Invitrogen ladder loaded. Only sufficient product seen for block 2. Indi= cates 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 previo= us 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 3= 0C

8/26

Start over the inductions, follow the igem protocol file for help. You w= ill need to make more LB. I have already made a few glycerol stocks. You wi= ll need to set up a 2mL innoculation in a culture tube as well as a 40mL in= noculation 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 weste= rn blot issue. Seems to be a lot of non-specific binding -we see blots of m= any 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 (ba= rely any) protein. Updated notebook, e-mailed Justin and Kevin for trouble = shooting.

BH- Type up lysing assay and bradford assay -give some specifics about w= estern 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 sol= ution 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. Not= e: 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.

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 ord= er to test lower gradient. Induction started at 2:30 am when OD600 of 40 mL= innoculations was 1.6(?)

9/2

Minimal Media

Ordered M9 minimal media 5X salts on Rust Lab tab. Should take till frid= ay 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 1= 00ng 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

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

Set up Gibson of BH002/3

9/3

Lysed cells after induction

Bradford Assay

Conducted Western blot

Ran colony PCR of TOPO reactions -only MC003 TOPO worked. BH002/3 Gib= sons worked, no TOPO results for MC002, BH002, BH003.

Inoculated negative control.

9/4/15

Set up Rhamnose time course experiment and rhamnose gradient experime= nt. Both used M9 media and LB media. M9 media arrived.

9/7/15

Miniprepped colony PCR for MC003, BH002, BH003.

9/8/15

Set up bradford of time-course and rhamnose gradient. Ran gel and tra= nsfer of assays. Ran negative vector control this time.

9/9

Finished western blot, imaged gel. Blotted only for KaiA and KaiC.

9/10

Set up induction again, be careful of transfer where errors seem to o= ccur. Blot for Kai B from previous assay.

Your Website Title

Like our team Facebook page, Genehackers@UChicago!

Questions? Comments? Send us an email!

@@ Line 19: / Line 19: @@
 <p>You can see what others teams have done to organize their notes:</p>
 <ul>
 <li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 <li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
@@ Line 25: / Line 25: @@
 <li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 </ul>
+<!-- autogen start -->
+<p><b>GeneHackers Summer 2015 Journal</b></p>
+<p><b>Week 1: </b></p>
+<p><b>Goals- take inventory, design constructs and primers, test competent =
+cells</b></p>
+<p>6/15/15 </p>
+<p>Worked on primer design quiz questions. Discussed shift of project direc=
+tion with Justin based on recently published paper (Chen, 2015): <u><a href=
+=3D"http://advances.sciencemag.org/content/1/5/e1500358.full" rel=3D"nofoll=
+ow">http://advances.sciencemag.org/content/1/5/e1500358.full</a></u></p>
+<p>Took inventory, created excel sheet online under <u>Protocols folder =E2=
+=80=9CGenehackers 2015 Inventory=E2=80=9D</u></p>
+<p>Downloaded SnapGene Viewer to work on plasmid constructs. </p>
+<p>6/16/15</p>
+<p>New idea for output system -create one construct with SasA/RpaA as activ=
+ator of output molecule, another with LabA/RpaA as inhibitor (negative feed=
+back loop) of output molecule. Based on article (Taniguchi, 2010). Started =
+design on constructs, possibly 5 in total:</p>
+<ol><li>Read-out activator (KaiCEE-RFP, RpaA, SasA) </li>
+<li>Read-out inhibitor (KaiCEA-RFP, RpaA, CikA)</li>
+<li>Test gene (kaibc promoter, GFP)</li>
+<li>Fusion SasA</li>
+<li>Fusion KaiC-P </li></ol>
+<p>Last two based heavily on paper (Chen, 2015)</p>
+<p>Met with Jennifer Moran -need to do safety training before autoclaving l=
+iquids and other procedures, will accomplish once more people are back.</p>
+<p><b>To Discuss: Is it worth having negative feedback regulator of RpaA/ o=
+utput molecule?</b></p>
+<p>6/17/15</p>
+<p>Decided to use CikA as negative regulator/inhibitor instead of Lab A. Ci=
+kA better characterized. Reviewed (Gutu. O=E2=80=99Shea, 2013). </p>
+<p>6/18/15</p>
+<p>Finished design on constructs 1, 2, 3 Deciding on RBS, perhaps need to u=
+se high efficiency promoters and lower efficiency RBS. Justin will email ka=
+ibc/Kai analog sequences. Decided on meeting 4pm Tuesday. </p>
+<p><b>To Discuss: Specific RBS and promoter strengths on different genes. <=
+/b></p>
+<p><b>Started Testing for Competent Cells</b></p>
+<p>*Used Justin/Rust Lab protocol instead of iGEM/team protocol because did=
+ not have cmrR plates. </p>
+<p>Labels:</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 s=
+ection </li>
+<li>Shake with beads and remove </li>
+<li>Incubate plate overnight 37C</li></ol>
+<p>6/19/15</p>
+<p><b>Checked on Transformed Plates</b></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 pla=
+ted))</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><b>Made 500 mL LB Solution </b></p>
+<p><b>Made CM plates</b></p>
+<p>	</p>
+<p>Temperature of Freezer=3D 6 C, Chloramphenicol storage temperature=3D 2-=
+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 Invent=
+ory) </p>
+<p>6/20/15</p>
+<p>Stock room closed :(, Will order things on Monday</p>
+<p>Placed cmR plates in left cold room, bottom right corner of room behind =
+some Rust plates </p>
+<p><b>Week 2: </b></p>
+<p><b>Goals- Finalize construct design, develop Competent Cells</b></p>
+<p>						6/22/15</p>
+<p>Worked on Week 1 Presentation</p>
+<p>Streaked 1 Tube of Comp 2014 Cells on LB Only  Plate for Competent E.col=
+i procedure -protocol under master list titled =E2=80=9CCompetent E.Coli 6/=
+/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>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 constru=
+ct worth it. Given that RpaA might have some basal phosphorylation level, i=
+nduced 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 b=
+io-brick. Final biobrick would possibly only have CikA, SasA. Overall conse=
+nsus 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 w=
+hich molecule want to consider as well as what is focus of experiment. Want=
+ KaiABC as submitted biobrick so perhaps focusing on biosynthesis pathway i=
+s 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 RP=
+I. </p>
+<p>Contact Danny for competent cells and Justin for Gibson primers</p>
+<p>6/24/15 </p>
+<p>Worked on designing primers. Contacted Danny for competent cell procedur=
+e, will complete on Friday. Will conduct both CaCl2 and RbCl2 procedures. A=
+lready have streaked LB only plate for competent cells in cold room. </p>
+<p>6/25/15 </p>
+<p>Finished designing primers, will check with Kevin tomorrow. Inoculated t=
+wo 5mL LB broths with 1-3 colonies each.  NEB Gibson Assembly Kit with comp=
+etent cells arrived! </p>
+<p>6/26/15 </p>
+<p>Carried out competent cell procedure. </p>
+<p> <u><a href=3D"http://www.unc.edu/depts/marzluff/Marzluff/Protocols_file=
+s/Preparation%20of%20Chemically%20Competent%20BL21%20or%20XL1%20blue%20usin=
+g%20rubidium%20chloride.pdf" rel=3D"nofollow">http://www.unc.edu/depts/marz=
+luff/Marzluff/Protocols_files/Preparation%20of%20Chemically%20Competent%20B=
+L21%20or%20XL1%20blue%20using%20rubidium%20chloride.pdf</a></u></p>
+<p>https://drive.google.com/open?id=3D0B7wkycR1BRlmWHQ5UGNOaW0xX25ndmE1aUxP=
+U01uclJCVmNV</p>
+<p>Used both Rust Lab and RbCl2 as a comparison. </p>
+<p><b>Week 3: </b></p>
+<p><b>Goals- Test efficiency of competent cells, start as much cloning as p=
+ossible</b></p>
+<p>6/29/15</p>
+<p>Conducted Transformation of CaCl2 and RbCl2. </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 w=
+hile 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>=3D (52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/20=
+uL plated))</p>
+<p>		      =3D 52 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>1.15 x 10^6 transf=
+ormants/ng (Rust)</u></p>
+<p>=3D52 cfus) / ( ((1 uL x 50 pg/uL x 1ng/1000pg)/1000uL soln))*((180/200 =
+uL plated))</p>
+<p>		      =3D 18 cfus/(4.5 x 10^-5) ng DNA plated =3D<u>4.00 x 10^5 transf=
+ormants/ng (RbCl2)</u></p>
+<p>6/30/15</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. A=
+lso 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 Osc=
+illation system were designed as well.</p>
+<p><b>Week 4: </b></p>
+<p><b>Goals- Order final gBlocks and primers. Standardize and develop speci=
+fic, in depth protocols. Practice Western Blots, start writing project repo=
+rt. </b></p>
+<p>7/20/15</p>
+<p>gBlocks for Oscillation and Read-Out systems were modified. See Dropbox =
+for final edits and modifications. </p>
+<p>7/21/15</p>
+<p>gBlocks for Oscillation and Read-Out systems were finally ordered. Prime=
+rs were designed. </p>
+<p>7/22/15</p>
+<p>Primers ordered. Reached out to grad advisers for Western Blotting techn=
+iques. Researched Gibson Assembly and Western Blot protocols. Will need to =
+research GFP Protocols. </p>
+<p>GFP Protocols</p>
+<p><a href=3D"http://advances.sciencemag.org/content/advances/1/5/e1500358.=
+full.pdf" rel=3D"nofollow">http://advances.sciencemag.org/content/advances/=
+/5/e1500358.full.pdf</a></p>
+<p>7/23</p>
+<p>Materials for practice western blot acquired.  Gibson Assembly protocol =
+drafted. </p>
+<p>7/24</p>
+<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>
+<p>7/27</p>
+<p>Primary and secondary antibody staining accomplished. Experiments more c=
+learly laid out. Need to start considering plan for pRha inducible promoter=
+ and how to alter stoichiometry.</p>
+<p><b>Week 5: </b></p>
+<p><b>Goals- Finalize and outline protocols, generate explanations for plas=
+mids and background info for wiki and presentation, order materials, gblock=
+ assembly</b></p>
+<p>7/28</p>
+<p>Western blot procedures expanded. See Aaron=E2=80=99s email about compat=
+ible backbones. This week discussed assays -will need to western blot for K=
+aiA before investigating oscillatory system in order to characterize input =
+L-Rhamnose to output Kai A production. Dilutions will occur on log scale fi=
+rst for L-Rhamnose. </p>
+<p>7/29</p>
+<p>Finished specific protocols -need to ask White lab for sonicator?. Looke=
+d up compatibility of backbones. pSB1,3,4 have pMB1 (copy number 100-300/ce=
+ll), p15A (low-medium 10-12 copy), pSC101 (~5 copies/cell). Will need to co=
+nstruct primers for kaibc/GFP onto the SasA+CikA/SasA plasmids. </p>
+<p>7/30/15</p>
+<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>
+<p>7/31/15</p>
+<p><b>Gibson Assembly</b></p>
+<p>5 uL of Gibson HiFi Master mix was used in each assembly reaction to min=
+imize 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 st=
+andardized amount 50 ng of largest block in each assembly. Assembled on ice=
+. Incubated on 50oC heatblock for 1 hour.</p>
+<p><img src=3D"cid:Image_0.png" /></p>
+<p><b>PCR</b></p>
+<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 o=
+f Master Mix with 1 uL from Gibson Assembly Mix.</p>
+<p>Recipe Phusion Master Mix:</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,=
+o for 1 min, 72o 7 min, Hold 4o</p>
+<p><b>Week 6: </b></p>
+<p><b>Goals- gblock assembly and Transformation</b></p>
+<p>8/3/15</p>
+<p><b>Decided on backbones:</b></p>
+<p><b>            	</b>Oscillator MC001 =E2=80=93 Cmr (standard igem backbo=
+ne for submitted biobrick)</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>Made 50 mL of 1% Agarose gel. General procedure: added agarose and 1X TA=
+E 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 1=
+-20 mins.</p>
+<p><b>Gel Electrophoresis</b></p>
+<p>Loaded 10uL of 6X loading dye into 50uL samples. Loaded 10 uL of 1 kB Pl=
+us 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 issu=
+e with evaporation in thermocycler. Gel too big for tray. Yields of product=
+s exist, however seems low. Issue with MC001 =E2=80=93no clear product visi=
+ble. PCR should be redone.</p>
+<p><b>Image:</b></p>
+<p><b>Gel Extraction</b></p>
+<p>Gel Weights:</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 volum=
+e of gel.</p>
+<p><b>PCR</b></p>
+<p>To redo PCR for MC001,4,5,6,7, 1X Master Mix for 15 reactions made. Two =
+reactions for each construct.</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 m=
+in, 72o 10 min, 4o hold.</p>
+<p>PCR Master mix also used for amplifying linear Cmr backbones (diluted in=
+uL, used 1 uL of sample for Phusion PCR).</p>
+<p><b>8/4/15</b></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 backbone=
+s. Products from MC004,5,6,7 and Cmr Linearized backbones extracted using g=
+el punches (borrowed from Rust Lab, need to order more to return). MC001 st=
+ill not very good yield. Next step to purify MC004-7 and linearized backbon=
+es, redo Gibson and PCR of MC001 using gradient thermocycler.</p>
+<p><b>Image:</b></p>
+<p><b>Gibson Assembly</b></p>
+<p>5 uL of Gibson HiFi Master mix, 1 uL PMC001_b1, 0.5965 uL PMC001_b2, 3.4=
+uL H2O, heatblock for 1 hour 50oC.</p>
+<p><b>PCR</b></p>
+<p>Master mix of 70 uL created (calculate ratio of 1X x 7/5)</p>
+<p>o   Phusion 5X HC Buffer =3D 14 uL</p>
+<p>o   dNTPs 10 mM  =3D 1.4  uL</p>
+<p>o   Phusion DNAP =3D 0.7 uL</p>
+<p>o   H2O =3D 43.4 uL</p>
+<p>o   DMSO =3D 2.1 uL</p>
+<p>o   DNA =3D1.4 uL</p>
+<p>o   F Primer MC003=3D 3.5 uL</p>
+<p>o   R Primer MC004 =3D3.5 uL</p>
+<p>10uL Master mix aliquoted into 7 samples.</p>
+<p>Thermocycler settings- 98o 2 min, 98o 15 s, 60o, 62o, 64o, 66o, 68o, 70o=
+, 72o 30 s, 72o 1 min, 72o 10 min, 4o hold.</p>
+<p>Actual Anneal temperatures- 60.0o, 62.0o, 63.3o, 66.6o, 68.2o, 69.7o, 72=
+.0o</p>
+<p>T=3D66.0o, G=3D6.0o for 30 cycles</p>
+<p><b>8/5/15</b></p>
+<p>Gel Electrophoresis</p>
+<p>Made 90 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye). Ru=
+n for 120V, 30 mins. EtBr cloud on gel seen, only ladder shows visible band=
+s. No other bands visible. Likely error with PCR and addition of EtBr.</p>
+<p><b>Image:</b></p>
+<p><b>Gel Extraction</b></p>
+<p>Used Promega spin columns/buffer to concentrate in 15 uL of DNA </p>
+<p>Purity Yields using nanodrop - C1 (Cam Backbone) -</p>
+<p>C2 (Cam Backbone) -58.9 ng/uL</p>
+<p>4 - 34.7</p>
+<p>4=E2=80=99 -</p>
+<p>5 - 31.5</p>
+<p>6 - 36.3</p>
+<p>7 - 40.1</p>
+<p><b> </b></p>
+<p><b>PCR</b></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>T=3D66.0o, G=3D6.0o for 30 cycles</p>
+<p><b>8/6/15</b></p>
+<p><b>Gel Electrophoresis</b></p>
+<p>Made 100 mL of 1% Agarose gel. Loaded 10uL of 7 samples(+loading dye) an=
+d 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 sho=
+ws faint products under 68,70, and 72o. Could mean issue with primers. Stra=
+ngely, no product of right gBlock size seen. Again could be primers.</p>
+<p><b>Image:</b></p>
+<p><b> </b><img src=3D"cid:Image_1.png" /></p>
+<p><b> </b></p>
+<p><b>Gibson Assembly:</b></p>
+<p>Assembled purified biobricks MC004, MC005, MC006, MC007 into Cam backbon=
+e. Used following recipe based on bps of insert and backbone. </p>
+<p><img src=3D"cid:Image_2.png" /></p>
+<p><b>Transformation of Assembled products:</b></p>
+<p>DNA straight from Gibson Assembly Reaction was transformed into competen=
+t cells. RbCl2 competent cells used. Efficiency of cells: <u>4.00 x 10^5 tr=
+ansformants/ng (RbCl2) </u></p>
+<ol><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 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 w=
+hile 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>*Negative control w/no transformed DNA resulted in 0 colonies. Negati=
+ve control set up on 8/7.</b></p>
+<p><b>PCR</b></p>
+<p>PCR of 8/3 Gibson PCR, 8/4 Gibson PCR conducted for further amplificatio=
+n. 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 e=
+ach PCR (5 samples in total). </p>
+<p>Added Ingredients to individual Samples</p>
+<p>o   Phusion 5X HC Buffer =3D 10 uL</p>
+<p>o   dNTPs 10 mM  =3D 1 uL</p>
+<p>o   Phusion DNAP =3D 0.5 uL</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>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><b>8/7/15</b></p>
+<p><b>Gel Electrophoresis</b></p>
+<p>1% Agarose gel run of PCR products from 8/6. Could not see products unde=
+r blue light. Under UV light, products seemed more specific. Still not as s=
+trong as in previous gels. Perhaps need to troubleshoot PCR better. </p>
+<p><b>Image:</b></p>
+<p><b>Transformation Results</b> </p>
+<p>Used iPhone application =E2=80=9CColonyCount=E2=80=9D to assist in count=
+ing plates.</p>
+<p>Plate Numbers are indicated on lower left of the pictures.</p>
+<p>Average is 321 colonies.</p>
+<p><img src=3D"cid:Image_3.jpg" /><img src=3D"cid:Image_4.jpg" /><img src=
+=3D"cid:Image_5.jpg" /><img src=3D"cid:Image_6.jpg" /></p>
+<p><b>Transformation Efficiency </b></p>
+<p>Used 1ul of 50pg/ul  of DNA </p>
+<p>4: 287 / 5*10^-5 =3D 5.74*10^6 cfu/ug</p>
+<p>5: 328 / 5*10^-5 =3D 6.56*10^6 cfu/ug</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><b>Primers</b></p>
+<p>Designed Sequencing primers as well as new primers for pMC001. Primers m=
+ade specifically for oscillator plasmid -overhangs incorporated to make pri=
+mers longer and more specific. </p>
+<p><b>PCR</b></p>
+<p>Prepared PCR of products seen on gel in morning (G1, G2, 001b1, 001b2, 0=
+b1, used 1 uL of leftover PCR reaction). Used Q5 High Fidelity polymerase=
+, as no Phusion available. </p>
+<p>Ingredients:</p>
+<p>	Q5 High-Fidelity 2X Master Mix- 25 uL</p>
+<p>	DNA -1 uL</p>
+<p>	F Primer -2.5 uL</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 f=
+or 2mins, hold at 4C</p>
+<p>30 cycles </p>
+<p>Prepared Colony PCR To confirm inserts of pMC004,5,6,7. Used Taq DNA pol=
+ymerase instead of phusion. </p>
+<ol><li>Pick single colony from plate, place in 50uL of dH2O (acts as DNA t=
+emplate)</li>
+<li>Add following PCR 1X Master Mix for Taq:</li></ol>
+<p>5 uL 10X buffer</p>
+<p>1 uL dNTPs</p>
+<p>1 uL 10 uM primer stock-VF2 and VR</p>
+<p>1 uL DNA stock</p>
+<p>0.5 uL Taq</p>
+<p>41.5 uL H2O </p>
+<p>Thermocycler Settings: 95C 2mins, 95C 15s, 55C 15s, 68C 45s (30 cycles),=
+C 10m, hold 4C</p>
+<p>-> Extend to 1min per kb (look up on product sheet)</p>
+<p><b>8/9/15</b></p>
+<p><b>Gel Electrophoresis-</b></p>
+<p>Ran 1% Agarose gel 120V, 30 mins. Ran both Colony PCR and Q5 PCR. 5uL ea=
+ch sample loaded. Different ladder used (Quick Load Purple 2-Log from NEB. =
+Same amount of EtBr (0.75 uL) used. </p>
+<p><b>8/10/15</b></p>
+<p><b>Gel Electrophoresis-</b></p>
+<p>Gel repeated, this time using leftover 45uL of sample. 1kB Plus invitrog=
+en ladder used. </p>
+<p><b>Innoculation-</b></p>
+<p>Due to failure of Colony PCR, colonies were inoculated and incubated. Wi=
+ll conduct direct miniprep on 8/11 and sequence to sequence. This should he=
+lp in determining if there is a problem with primers/insert or with the PCR=
+. </p>
+<p><b>Gibson Assembly-</b></p>
+<p>Gibson assembly of BH001_b1 and Cam backbone conducted. </p>
+<p><b>PCR-</b></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 t=
+emplate DNA used. </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% Agar=
+ose</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 promote=
+r/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  G=
+ibson</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>Thermocycler settings- 98C 2min, 98C 15s, 67C 30s, 72C 1.5 mins, 72C 10m=
+in, 4 hold. </p>
+<p><b>8/11/15</b></p>
+<p><b>Gel Electrophoresis-</b></p>
+<p>2% gel for Terminator cloning sample and 1% gel for other PCR samples (s=
+ee table) run. 120V for 30 min. </p>
+<p><b>Miniprep</b></p>
+<p><b>BH protocol</b></p>
+<p><b>PCR</b></p>
+<p><b>	</b><u>PCR of Minipreps</u></p>
+<p>(BH protocol)</p>
+<p>	PCR of MC002/3 blocks</p>
+<p>Phusion 7 x of 1X mix</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 D=
+NA.</p>
+<p><b>DNA Template</b></p><p><b>Primers</b></p><p>MC002_b1</p><p>MC029, MC0=
+</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>Thermocycler Settings-</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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 (=
+s x 1.8kb -largest product)</p><p>Final Extension</p><p>72=C2=B0C</p><p>1=
+min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+<p>Thermocycler Settings for Mini-Prep PCR-</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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 Exten=
+sion</p><p>72=C2=B0C</p><p>5 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+<p><b>8/12/15</b></p>
+<p>PCR of Terminator </p>
+<p>2 50 uL 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>	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, 6=
+C for 30s, 72C for 2 min 30 cycles: 98C for 15s, 72C for 1.5 min, 1 cycle =
+C for 10 min, 4C hold. </p>
+<p><b>Gel Electrophoresis-</b></p>
+<p>50 mL 2% gel, 150 mL 1% gel, and 100 mL 1% gel run for Miniprep PCR as w=
+ell as MC002/3 clone parts PCR. 25 uL of sample loaded for Terminator 2%  g=
+el. 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 4=
+,43,51,52,53,54,61,62,63,64,74 for sequencing. </p>
+<p><b>Images:</b></p>
+<p><b>Gel Purification-</b></p>
+<p>Extracted correctly sized product fragmnets under blue light: T-189 bps,=
+ GFP 1249 bps. </p>
+<p>Weights of gels:</p>
+<p>GFP 1-63.8 mg</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>
+<ol><li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+<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. </l=
+i>
+<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 3=
+s</li>
+<li>Nanodrop (use EB buffer as blank, load 1.5 uL sample)</li></ol>
+<p>Purity recorded w/Nanodrop:</p>
+<p>T1- 5.1ng/uL 260/280-14.84</p>
+<p>T2-23.1ng/uL 260/280-1.92</p>
+<p>GFP 1- 41.6ng/uL 260/280-1.94</p>
+<p>GFP 2- 33.1ng/uL 260/280-2.03</p>
+<p><b>Plan for MC002_b1, MC003_b1: </b>There is low yield of insert probabl=
+y due to repeating regions of DNA in blocks 1 of MC002 and MC003. As IDT ex=
+pressed, there are a lot of low mass products that are more efficiency ampl=
+ified by primers. Therefore, as Jennifer Moran mentioned, the best course o=
+f action would be to insert these two gblocks into a Zero Blunt TOPO PCR Ve=
+ctor and transforming into competent cells to amplify our gblocks. After pr=
+oducing colonies, we can PCR and then screen for colonies with correct prod=
+uct size. We will then miniprep and send these blocks in for sequencing. Th=
+is will take more time, but will ensure the purity of the insert. After con=
+firming the correct sequence, the DNA from the miniprep/gel extraction? can=
+ be used to gibson the GFP/kaibc, the terminator, and the first blocks toge=
+ther. </p>
+<p><b>Mini-Prep Results (Nano-Drop)</b></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>5=
+</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.=
+</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>=
+.3</p><p>74</p><p>1.92</p><p>1.92</p><p>53.3</p>
+<p>The highlighted ones are the ones that we choose to sequence.</p>
+<p><b>Sequencing-</b></p>
+<p>The concentration of the DNA templates were too low, so we used 10 ug of=
+ each.</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>MC003 (F) [2]</p>
+<p>MC041 (F) [3]</p>
+<p>MC023 (F) [4]</p>
+<p>MC022 (R) [5]</p>
+<p>VR [6]</p>
+<p>[41]</p>
+<p>[43]</p>
+<p>[53]</p>
+<p>[54]</p>
+<p>[62]</p>
+<p>[63]</p>
+<p>[71]</p>
+<p>[73]</p>
+<p><img src=3D"cid:Image_7.png" /></p>
+<p>^Things sent.</p>
+<p><b>Transformation of BH001:</b></p>
+<p>DNA was transformed into competent cells. RbCl2 competent cells used. Ef=
+ficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></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 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 w=
+hile 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>8/13/15-</b></p>
+<p><img src=3D"cid:Image_8.png" /></p>
+<p>Transformation Efficiency of BH001-</p>
+<p>amt dna used/plate</p>
+<p>Plates 1 and 2 were discarded, a colony PCR was done on 6 samples from P=
+late 3</p>
+<p>They were PCRed separately with two different annealing temperatures, 61=
+.6 and 66. </p>
+<p><b>Gibson Assembly</b></p>
+<p>Gibson assembly using 5uL of gibson master mix 2x conducted. Assembled o=
+n ice. Incubated for 1 hour 50C heatblock.</p>
+<p><b>PCR-</b></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><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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>Thermocycler Settings for Mini-Prep PCR- Low Temp</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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 Exten=
+sion</p><p>72=C2=B0C</p><p>5 min</p><p>Hold</p><p>4=C2=B0C</p><p> Hold</p>
+<p>Thermocycler Settings for Mini-Prep PCR- High Temp</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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>
+<p><b>Gel Electrophoresis-</b></p>
+<p><b>	</b>100 mL of 1% agarose gel run 120V, 30 mins. Samples loaded inclu=
+de 45 uL of BH003 biobrick and 20 uL of colony PCRs. 5 uL 1 kB plus Invitro=
+gen 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>
+<p><b>Image-</b></p>
+<p><b>Gel Extraction-</b></p>
+<p>BH003 biobrick extracted from gel. Machery-Nagel protocol used for gel e=
+xtraction. </p>
+<p>Weight of gel: 85.2 mg</p>
+<p>Used Machery-Nagel clean up method:</p>
+<li>Add 200uL NTI Binding buffer / 100 mg gel </li>
+<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. </l=
+i>
+<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 3=
+s</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><b>PCR-</b></p>
+<p><b>	</b>Made 4 1X 50uL samples to PCR ampicillin backbone. Used 1 uL of =
+ng/uL amp linearized backbone. Primers MC001 and MC002. </p>
+<p>PCR Master Mix Recipe-</p>
+<p>o   Phusion 5X HF Buffer =3D 50 uL</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><b>Gibson Assembly-</b></p>
+<p><b>	</b>Given success of gibson and transformation with BH001, decided t=
+o try assemble pMC001 and transform directly. Reaction incubated on heatblo=
+ck for 1 hour at 50C. </p>
+<p><img src=3D"cid:Image_9.png" /></p>
+<p>	</p>
+<p><b>Gel Electrophoresis-</b></p>
+<p><b>	</b>50 mL of 1% agarose gel made to run amp backbone amplifications.=
+.5 uL EtBr used. Gel run 120V for 30 mins. 1 kB Plus Invitrogen ladder us=
+ed. 45 uL of samples loaded. </p>
+<p><b>Image- </b></p>
+<p><b>Gel Extraction and Purification-</b></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>A1 - 32.3 ng/uL, 260/280- 1.84</p>
+<p>A2 - 33.4 ng/uL, 260/280- 1.59 </p>
+<p><b>Gibson-</b></p>
+<p>Gibson assembly of ampicillin backbone to BH003 insert conducted. Used 5=
+uL of gibson master mix 2x conducted. Assembled on ice. Incubated for 1 hou=
+r 50C heatblock.</p>
+<p><b>Transformation of BH003:</b></p>
+<p>DNA was transformed into competent cells. RbCl2 competent cells used. Ef=
+ficiency of cells: <u>4.00 x 10^5 transformants/ng (RbCl2) </u></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 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 w=
+hile 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><b>Transformation-</b></p>
+<p>	MC001 and BH003 gave good results from transformation. Colonies were sm=
+all, but evenly distributed. Colonies could be small because plates incubat=
+ed late last night. </p>
+<p><b>Colony PCR of BH003/MC001-</b></p>
+<p>Diluted one colony from plates with most growth into 50uL of dH2O to ser=
+ve as DNA template. </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><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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 m=
+ins</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>Sequencing Primers for MC001- MC003,MC031,MC032,MC006,MC033,=
+ MC008</blockquote></p>
+<p><b>8/15/</b></p>
+<p><b>Gel Electrophoresis </b></p>
+<p><b>	</b>1% Agarose gel run for colony PCR. 1 kB Invitrogen plus ladder u=
+sed. 50 uL of sample loaded. </p>
+<p><b>8/17</b></p>
+<p><b>Sequences Submitted</b></p>
+<p>MC001 note submitted on weekend for sequencing. MC001 submitted in morni=
+ng 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>VF2 (1)</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>Colony PCR of MC001 set up again as upon closer examination, insert seem=
+s too small to be correct. </p>
+<p>Diluted one colony from plates with most growth into 50uL of dH2O to ser=
+ve as DNA template. </p>
+<p>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2=
+ uL DNA):</p>
+<p>	HF Buffer: 18.00</p>
+<p>	DMSO: 2.70 uL</p>
+<p>	dNTPS: 1.80</p>
+<p>	H2O: 55.</p>
+<p>	DNAP: 4.25 uL</p>
+<p>	Used 4.50 uL of 10X MC003 and MC004 for mix. </p>
+<p>Thermocycler Settings for Colony </p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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 m=
+ins</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><b>8/18</b></p>
+<p><b>Gel Electrophoresis-</b></p>
+<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 res=
+ults from colony PCR, indicating something wrong during PCR. Likely issue w=
+ith 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 eva=
+poration in thermocycler, which is why samples 115,116,117,118,128 could no=
+t be loaded. </p>
+<p><b>Image-</b></p>
+<p><b>Sequencing Results</b></p>
+<p>Sequencing successful for MC004,5,7. MC006 and MC007 samples probably mi=
+slabelled as sequencing indicates correct MC006 phosphomimetic is in MC007 =
+sequence and vice versa. Otherwise, MC007,5, and 4 are all correctly sequen=
+ced. Point mutation in MC006 (labelled MC007) samples base 244 of Snapgene =
+file. Point mutation seems to be silent (GGC to GGT, translate features lis=
+ts both as glycine. Four read out KaiC variants seem good to go. </p>
+<p>MC001 -still waiting for colony PCR/ specific primers to arrive for asse=
+mbly</p>
+<p>MC002/3 -still waiting for TOPO kit to come for transformation ligation =
+and assembly</p>
+<p>MC004/7 -all assembled </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><b>	</b>We will be ligating and transforming four samples in order to al=
+low the bacteria to amplify blocks 1 of MC002 and MC003. </p>
+<ul class=3D"small"><li>MC002_b1</li></ul>
+<li>MC003_b1</li>
+<li>MC002_b1 after PCR</li>
+<li>MC003_b1 after PCR </li></ul>
+<p><blockquote>Ligation</blockquote></p>
+<ol><li>Combine 2uL product, 1 uL provided salt solution from pTOPO kit, 2u=
+L H20, 1 uL pCR II-Blunt-TOPO vector </li>
+<li>Pipette up and down a few times to mix. </li>
+<li>Incubate 5 min at room temperature.   </li>
+<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>
+<ol><li>Thaw one vial of Chemically Competent E. coli cells per transformat=
+ion on ice. </li>
+<li>Add 2=CE=BCl ligation product to the thawed cells.  Keep on ice for 30 =
+mins. </li>
+<li>Transfer vials to a 42=C2=B0C water bath for 45 seconds. Immediately re=
+turn 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> pTOPO kit) </p>
+<p> <i>  Note: if you do not have a sufficient number of tranformants in 20=
+=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 su=
+pernatant, resuspend the cells by pipeting up and down.</i></p>
+<p><b>PCR</b></p>
+<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>
+<p>	Set up 2 50uL 1X PCR Reactions:</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><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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><b>Innoculation </b></p>
+<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>
+<ol><li>Aliquot enough LB for 2mL/sample into a Falcon tube. Pipette 2mL of=
+ LB into each culture tube/sample. </li>
+<li>Pipette all of the colony suspension into the culture tube as well. </l=
+i>
+<li>Put in antibiotic to act as primary screening for insert -Cam 1uL, Amp =
+uL (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><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 produ=
+ct size conducted under blue light. 5uL 1kB Plus Invitrogen ladder loaded. =
+uL of samples loaded. </p>
+<p><b>Image</b></p>
+<p><b>8/19/15</b></p>
+<p><b>Transformation Results</b></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 i=
+f cells are very efficient as cells could also be very conducive to transfo=
+rmation. </p>
+<p><b>Sequencing Results</b></p>
+<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>
+<p><b>Miniprep-</b></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 a=
+ll 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 c=
+apped 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 min=
+s. </li>
+<li>Load supernatant (750 uL) into Spin column (machery nagel used) and col=
+lection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow thro=
+ugh 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. Di=
+scard 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 D=
+NA. Store at 4C short term, -20C long term. </li></ol>
+<p>Nanodrop results-</p>
+<p>115: 91.8 ng/uL, 260/280: 1.90</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>
+<p><b>PCR </b></p>
+<p><b>	</b>Set up PCR for both miniprep PCR and PCR of MC002_b1 and MC003_b=
+to redo ligation and transformation. </p>
+<p>Set up 2 50uL 1X PCR Reactions for MC002_b1 and MC003_b1:</p>
+<li>dNTPS: 1uL</li>
+<li>H2O: 31 uL</li>
+<li>DNAP: 0.5 uL</li>
+<li>DNA: 1 uL (gblocks MC002_b1 and MC003_b1)</li>
+<li>DMSO: 1.5uL </li>
+<li>HF Buffer: 10uL</li>
+<li>F Primer: 2.5uL (MC029)</li>
+<li>R Primer: 2.5 uL (MC010 for MC002_b1 and MC014 for MC003_b1)</li></ul>
+<p>Made 9/5x of 1X PCR Master Mix for 10uL samples (used 9.8 uL Mix and 0.2=
+ uL DNA):</p>
+<p>	HF Buffer: 18.00</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 ot=
+her for 50uL samples)-</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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>1% agarose gel run at 120V for 30 mins. Loaded 10uL and 45 uL for minipr=
+eps and MC002/3 gel extractions respectively. Loaded 5uL of 1kB Plus Invitr=
+ogen ladder. </p>
+<p><b>Image- </b></p>
+<p><img src=3D"cid:Image_10.png" /></p>
+<p><b>8/20</b></p>
+<p><b>Sequences Submitted</b></p>
+<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 s=
+ubmitted with 10uL of 4uM primers. Samples 115,117, 125, 127, 128 sent. </p=
+>
+<p>Primers used: </p>
+<p>VF2 (1)</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>New primers for MC001 arrived. Proceeded with Gibson assesmbly. Incubate=
+d mix at 50C for 1 hour.</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 u=
+sed 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>	HF Buffer -75.00 uL</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>DNA : Primers</p>
+<p>MC002_b1 : MC029, MC010</p>
+<p>MC003_b1 : MC029, MC014</p>
+<p>Gibson rxn : 1b1F, 1b2R</p>
+<p>MC001_b1 : 1b1F, 1b1R</p>
+<p>MC002_b2:  1b2F, 1b2R </p>
+<p>Thermocycler settings:</p>
+<p>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and ot=
+her for 50uL samples)-</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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>
+<p><b>Gel Electrohporesis-</b></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 extracte=
+d separately. </p>
+<p><b>8/21</b></p>
+<p><b>PCR</b></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>o   Phusion 5X HF Buffer =3D 15 uL</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><b>	</b>MC001_b1 and MC001_b2 extracted yesterday were purified. Used Ma=
+chery-Nagel protocol. </p>
+<p>Weights-</p>
+<p>1b1 -13.4 mg</p>
+<p>1b2 - 133.4 mg</p>
+<p>Nanodrop results-</p>
+<p>1b1 - 13.1 ng/uL</p>
+<p>1b2 -19.2 ng/ul</p>
+<p><b>Gel Electrophoresis</b></p>
+<p><b>	</b>1% agarose gel run for gradient PCR. 120V for 30 mins. 1kB Invit=
+rogen ladder used. 10uL of sample loaded. Expected product size ~3000 bps. =
+No expected product size appeared on gel. Perhaps indicates issues with Gib=
+son assembly.  24 inoculated colony PCRs also run on separate gel. 3 coloni=
+es chosen for inoculation and miniprep: 3,9 and 22. </p>
+<p><b>Image-</b></p>
+<p><img src=3D"cid:Image_11.png" /></p>
+<p><img src=3D"cid:Image_12.png" /></p>
+<p><b>Gibson- </b></p>
+<p><b>	</b>Gibson of MC001 repeated. Incubated mix at 50C for 1 hour. </p>
+<p><b>PCR- </b></p>
+<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>
+<p>	DMAP: 0.5 uL</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 (MC00=
+_b2_F_new, MC001_b2_R_new), MC001 Gibson (MC001_b1_F_new, MC001_b2_R_new)<=
+/p>
+<p>PCR Conducted one at high anneal temperature (68C) one at low anneal tem=
+perature (64C)</p>
+<p>Thermocycler Settings (used 2 thermocyclers, one for 10uL samples and ot=
+her for 50uL samples)-</p>
+<p><b>STEP </b></p><p><b>TEMP </b></p><p><b>TIME </b></p><p>Initial Denatur=
+ation</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>
+<p><b>	</b></p>
+<p><b>Gel Electrophoresis-</b></p>
+<p><b>	</b>1% agarose gel run at 120V for 30 mins. 1kB Plus invitrogen ladd=
+er used. 50uL of samples loaded. Results very inconclusive. </p>
+<p><b>Image-</b></p>
+<p><b>8/24</b></p>
+<p><b>Miniprep-</b></p>
+<p>	Inoculated TOPO MC002_b1 and MC003_b1 samples miniprepped using invitro=
+gen protocol. </p>
+<p>Miniprep inoculations 115,116,117,118,125,126,127,128.</p>
+<li>Centrifuge cells at 12000 rcf for 3 mins to harvest cells. Remove all m=
+edium. </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 c=
+apped 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 min=
+s. </li>
+<li>Load supernatant (750 uL) into Spin column (machery nagel used) and col=
+lection tube. Centrifuge column at 12,000 rcfs for 1 min. Discard flow thro=
+ugh 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. Di=
+scard 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 D=
+NA. Store at 4C short term, -20C long term. </li></ol>
+<p>Nanodrop results-</p>
+<p>A21: </p>
+<p>A22: </p>
+<p>A31:</p>
+<p>A32: </p>
+<p>B21: </p>
+<p>B22: </p>
+<p>B31: </p>
+<p>B32: </p>
+<p>None were sufficient enough to send for sequencing. Likely errors also d=
+ue to overgrowth of colonies. </p>
+<p><b>PCR </b></p>
+<p>Gradient PCR of only MC001 blocks conducted. </p>
+<p>Master mix of 75 uL created (calculate ratio of 1X x 7.5/5)</p>
+<p>o   Phusion 5X HF Buffer =3D 37.5 uL</p>
+<p>o   dNTPs 10 mM  =3D 3.75  uL</p>
+<p>o   Phusion DNAP =3D 1.875 uL</p>
+<p>o   H2O =3D 116.25 uL</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>1 % agarose gel run for 30 mins at 120V. 25 uL of samples loaded. 1kB pl=
+us Invitrogen ladder loaded. Only sufficient product seen for block 2. Indi=
+cates issues with block 1. </p>
+<p><b>Image</b></p>
+<p><img src=3D"cid:Image_13.png" /></p>
+<p><b>8/25</b></p>
+<p>Sequencing Results: Found a sample (127) containing entire MC001 insert!=
+ </p>
+<p>Innoculated sample 127 and prepared for induction with L-Rhamnose. </p>
+<p>Re-plated TOPO reactions for MC002/3 as overgrowth of colonies on previo=
+us 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 3=
+C </p>
+<p><b>8/26</b></p>
+<p>Start over the inductions, follow the igem protocol file for help. You w=
+ill need to make more LB. I have already made a few glycerol stocks. You wi=
+ll need to set up a 2mL innoculation in a culture tube as well as a 40mL in=
+noculation in a flask. The 40mL innoculation will be used for inductions. [=
+Finished]</p>
+<p>Redo TOPO transformations. </p>
+<p><b>8/27</b></p>
+<p><b>8/28</b></p>
+<p><b>8/31</b></p>
+<p>Reviewed work done over past three days. Discussed ways to resolve weste=
+rn blot issue. Seems to be a lot of non-specific binding -we see blots of m=
+any 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 (ba=
+rely any) protein. Updated notebook, e-mailed Justin and Kevin for trouble =
+shooting. </p>
+<p>BH- Type up lysing assay and bradford assay -give some specifics about w=
+estern blot (how much each sample, and calculations) </p>
+<p>E-mail sleep people</p>
+<p><b>9/1</b></p>
+<p><b>Induction Solutions</b></p>
+<p>In order to re-do induction western blots, made new 10% Rhamnose w/v sol=
+ution by diluting 1 g of Rhamnose in 9 mL of water to bring final volume to=
+mL.</p>
+<p><b>Culture</b></p>
+<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. Not=
+e: 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>
+<p><b>Dilutions of Rhamnose Inducer Samples</b></p>
+<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 ord=
+er to test lower gradient. Induction started at 2:30 am when OD600 of 40 mL=
+ innoculations was 1.6(?) </p>
+<p><b>9/2</b></p>
+<p><b>Minimal Media</b></p>
+<p>Ordered M9 minimal media 5X salts on Rust Lab tab. Should take till frid=
+ay to arrive. </p>
+<p><b>Restriction Digest </b></p>
+<p>Protocol from iGEM.</p>
+<h2>Digest</h2>
+<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 1=
+ng 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><b>Transformed new TOPO reactions for MC02/3 and BH002/3</b></p>
+<p><b>Set up Gibson of BH002/3</b></p>
+<p><b>9/3 </b></p>
+<p><b>Lysed cells after induction </b></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 Gib=
+sons 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><b>Set up Rhamnose time course experiment and rhamnose gradient experime=
+nt. Both used M9 media and LB media. M9 media arrived. </b></p>
+<p><b>9/7/15</b></p>
+<p><b>Miniprepped colony PCR for MC003, BH002, BH003. </b></p>
+<p><b>9/8/15</b></p>
+<p><b>Set up bradford of time-course and rhamnose gradient. Ran gel and tra=
+nsfer of assays. Ran negative vector control this time. </b></p>
+<p><b>9/9</b></p>
+<p><b>Finished western blot, imaged gel. Blotted only for KaiA and KaiC. </=
+b></p>
+<p><b>9/10</b></p>
+<p><b>Set up induction again, be careful of transfer where errors seem to o=
+ccur. Blot for Kai B from previous assay. </b></p>
+<!-- autogen end -->
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