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Team:Stanford/Notebook/Lab Work/Week 5
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#B1006: cut with XbaI and SpeI | #B1006: cut with XbaI and SpeI | ||
#4C5 backbone: cut with EcoRI, PstI | #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) | ||
==7/30 Friday== | ==7/30 Friday== | ||
Revision as of 17:50, 4 August 2010
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Spring: Brainstorming | Spring Meetings
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|
7/26 Monday
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
- 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)
