Team:Stanford/Notebook/Lab Work/Week 5

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7/26 Monday

Alex's Notebook

Get picture from gel imager. Run the gel. Gel extract. Ligate. Transform. Plate. Both BW and DH5alpha. Order:  Heat shock cells  EcoRI-HF  T4 Ligase  MinElute tubes (?)

PCR: RBS’s  100 ng template  .5 ul primers (200 nM). Also try 2 (800 nM).

Promoters Try varying concentrations for NC and C  20 uM primer solution, 50 ul tot. vol. Want: 100, 200, 300, 400, 500 nM conc.  Use: .25, .5, .75, 1, and 1.25 uL primers and 4.5, 4, 3.5, 3, and 2.5 uL H2O.  Try 200, 400, 1000 (2.5 uL), and 2000 (5 uL) first

Try 2+3, then +1+4, one PCR purified and other not.  20 uM primer solution, 50 ul tot. vol.

 40 sm, 2+3, PCR purify, transfer. 5 uL each primer. SHOULD NOT WORK!!!  40 sm, 2+3, direct transfer.  40 sm, 1+4+2+3. 4 uL primer 1, 4 uL primer 4, and 2 of template (transfer).  40 sm, 1+4+2+3. 4 uL primer 1, 4 uL primer 4, and 2 of template (transfer).

 45 sm, 2+3, PCR purify, transfer. 2.5 uL each primer.  45 sm, 2+3, direct transfer.  45 sm, 1+4+2+3. 2 uL primer 1, 2 uL primer 4, and 1 of template (transfer).  45 sm, 1+4+2+3. 2 uL primer 1, 2 uL primer 4, and 1 of template (transfer).

 From best of these reactions, take .5 of P1 and P4 with .1 PCR purified template.  Use YA, 30 cycles.

Laura's Notebook

helped Francisco aliquot competent cells (thaw main tube- 1 mL- on ice, transfer 50 uL to each of 20 pre-chilled 0.5 mL microfuge tubes)

7/27 Tuesday

Laura's notebook

redo failed ligation: vary vector/insert ratios

	prior recipe	ligation 1	ligation 2	ligation 3
water	none	11.0	7.0	none
vector	5.0	2.0	2.0	2.0
insert	12.0	4.0	8.0	15.0
10X buffer	2.0	2.0	2.0	2.0
ligase	1.0	1.0	1.0	1.0

• vector=B1006 linearized (cut with EcoRI, XbaI; inserts=GFP or RFP cut with EcoRI, SpeI (6 ligations set up)
• Francisco transformed these

7/28 Wednesday

Laura's Lab Notebook

miniprepped (ProMega kit): GFP and B1006 terminator (RFP didn't grow) NanoDrop data:

sample	260/280	260/230	ng/uL
B1006	1.61	0.95	49.4
GFP	1.83	1.22	96.4

ran 2% diagnostic gel, 75V, 1 hour

• order: 100bp ladder, RSID1C PCR product, GFP digestion, RFP digestion, terminator digestion
• all samples: 1 uL sample + 1 uL loading dye

ran 2% gel for gel extraction, 75V, 1 hour

• order: 100bp ladder, GFP digestion, RFP digestion
• 10 uL loading dye added to 50 uL digestions, 40 uL loaded on gel

gel extraction of GFP, RFP digestion products (Qiagen kit)

	tube (g)	tube + slice (g)	gel slice (g)	uL QG
GFP	1.01	1.03	0.02	60
RFP	1.01	1.03	0.02	60

ran 2% diagnostic gel of gel purified GFP and RFP digests

• order: 100bp ladder, GFP, RFP
• 1 uL sample + 1 uL dye + 4 uL H2O

B1006 terminator NanoDrop data (diluted 50:50 with H2O)

260/280	260/230	ng/uL
1.47	1.39	10.4

• = 20.8 ng/uL in original sample

Digest concentrations still very low... set up larger overnight cultures- 9 mL cultures, 2 cultures each part:

• B1006 (terminator)
• GFP
• RFP
• pBAD (I0500)
• pLUX (F2620)

7/29 Thursday

Laura's Notebook

Miniprepped (ProMega kit): RFP, GFP, B1006 terminator, F2620 (pBAD didn't grow)

• all 18 mL (2- 9mL cultures) combined into one miniprep for each
• Francisco did nanodrop: he has concentration data

set up digestions for both types of ligations (two-part and 3a protocol)

1. RFP and GFP: cut with EcoRI, SpeI (2 digestions each)
2. B1006 terminator: cut with EcoRI 2 hours, heat kill 80oC 20 minutes, then add XbaI overnight
3. B1006: cut with XbaI and SpeI
4. 4C5 backbone: cut with EcoRI, PstI

• for two part ligations: use GFP or RFP from #1, terminator from #2 (2 ligations- one for GFP, one for RFP)
• for 3a ligation: use GFP or RFP from #1, terminator from #3, 4C5 backbone from #4 (2 ligations- one for GFP, one for RFP)

Meeting with Rayka- suggestions for Friday's (tomorrow's) meeting:

• include a brief review/overview of project for clarification
  • basic schematics of the two sides of the project
  • details of each, including the devices we plan to build
  • ligation schemes in visuals
  • for digital, analog: 1. overall plan 2. ligation scheme (home slide) 3. troubleshooting/challenges (including potential solutions)

suggestion for ligations: add ATP (is most likely to go bad in buffer) include plans for next week: minimum and ideal agenda again after lab stuff Twitter slide: change title to Gold Medal Requirements (add Facebook?) Lab Logistics: suggestions to collaborate in lab more efficiently

7/30 Friday

Laura's Notebook

9am iGEM meeting

in attendance: team (Chris, Karina, Alex, Francisco, Laura), Christina, Ryan, Rayka, Anusuya, Graham

Things to accomplish/address by next week:

• potential names of circuits (Laura, Rayka)
• flow cytometry: Does Scanford work for E. Coli? (Chris, Graham)
• get ligations to work! (everyone)
  • make sure DNA concentration is high enough
  • when looking at gel to estimate concentration, look directly at gel (not at image on computer screen)
  • use fresh ligase buffer (or add ATP)
  • get successful ligation to use as positive control (from Ryan?)
• get RSID1C PCR working
  • check concentration of primers: mix well, make new working stock
  • double check annealing temperatures, correct sequences
• 4 piece PCR (Alex)- order as 2 pieces?
• circuit diagrams, including levels of abstraction, consistent formats
• Twitter:
  • list/group
  • record audio (in as many languages as possible)