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
Alex's Notebook
Continue w/ PCR. 100 seems okay. Nothing from 107, since sequence is messed up. I5 and F26 look ok. I0500 (1255 bp) Good F2620 (1106 bp) Good enough.
J100 (98 vs 62 bp) Good enough. J107 (98 bp) Nothing.
AfsS C (123 bp) Good AfsS NC (104 bp) Good
Next steps: PCR purify (regular and MinElute). Nanodrop. RD: E/P. Spacer should be enough. Heat inactivate (80 C for 20 min, 75 C for 20 min for Klenow), RD cleanup. Nanodrop. Ligate. Ratios: 20 uL total: 1 uL ligase, 2 uL buffer, 8 uL, 8 uL insert, 375 ng vector. Heat inactivate (65 C for 20 min.) and transform.
New scheme for AfsS assembly: 1+2, 3+4, gel electrophoresis to get rid of primers. Use -------------- for primer concentration. Gel extract wanted product. Mix in a single reaction. Use same as above.
Check plates. Some grew. Culture O/N and do colony PCR. Focus on parts needed. RD today. All parts. Get extra RD enzyme (BglI). Control: 1A2 @ EcoRI, and use blunting enzyme from Ryan. 100 uL total: 24 uL DNA, 52 uL H2O, 10 uL BSA, 10 uL NEB buffer (check which to use), 4 uL enzymes. Gel electrophoresis and extract. Ligate and heat inactivate (as above). Transform. Use brown stripe or use new ligase buffer. NEED MORE LIGASE!!! 1.5 uL DNA, incubate on ice.
Prepare more electro-competent cells. Use DH5 alpha 21. Check for space in the incubator.
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
Alex's Notebook
Cultivate cells. PCR 1,2 and 3,4. Gel, gel extract.
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
Alex's Notebook
Transform using ccdb vector w/ new ligase and ligase buffer. Final PCR assembly 1,2+3,4. Failure.
Karina's Notebook
Goal: We want to try BioBrick 3A Free Assembly! [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf Protocol found here.]
Miniprep DNA
Helped Chris miniprep plasmids that contain ccdb to use for 3A assembly. Used [http://www.promega.com/tbs/tb225/tb225.pdf Wizard Plus Protocol] (note: Procedure takes the whole morning!)
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)
- RFP and GFP: cut with EcoRI, SpeI (2 digestions each)
- B1006 terminator: cut with EcoRI 2 hours, heat kill 80oC 20 minutes, then add XbaI overnight
- B1006: cut with XbaI and SpeI
- 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
Alex's Notebook
Colony PCR: 1. sG 2. FsG 3. I5E 4. B34 + J100. 1A2: 78 bp + 120 bp = 198 bp 3C5: 115 bp + = bp
Diagnostic of PCR and RD products. RD: 3 samples, ~350 ng/uL 1. B34 2. J100 3. J119
PCR: 6 samples, ~48, ~50, ~48, ~16, ~42, ~32. 1. C 2. C 3. NC 4. F26 5. I5 6. J100
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)