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Team:Stanford/Notebook/Lab Work/Week 6
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8/2 Monday
Greg's Notebook
- Inoculated freezer stocks of most of our parts with Alex
- Began planning for promoter characterization project:
- Going to do 3A ligations of the form: promoter + superfolded GFP + Kan plasmid
- Process:
- Digest parts with at least 2 ug DNA
- PCR cleanup
- Diagnostic gel (remember to save uncut DNA for control)
- Ligate in PCR tubes
- Heat inactivate at 65 C for 20 min
- Performed first digestion (F2620 + sfGFP + pSB4k5)
| Part # | Part amount | BSA | NEB (#, amount) | Enzymes (1 uL each) | Water | F2620 | 10.8 | 3 | 2, 3 | E + S | 11.2 | pSB4k5 | 20 | 3 | 3, 3 | E + P | 2 | sfGFP | 11.76 | 3 | 2, 3 | X + P | 10.24 |
- Let digestions run for 1 hour at 37 C
- Ran diagnostic gel:
- sfGFP digested nicely, F2620 yielded 3 bands corresponding to insert, cut plasmid, and uncut plamid
- pSB4k5 gave only one band and a smudge, so I decided to redigest with only .5 uL of each enzyme, leaving it to run overnight
8/3 Tuesday
Greg's Notebook
- Ran diagnostic gel of overnight pSB4k5 digest
- Still didn't work; there were 3 bands, the smallest of which was about the size of the plasmid backbone, and there was no band corresponding to the ccdB insert
- Decided to re-redigest pSB4k5 using .5 uL of each enzyme for only 1 hour
- After running on a diagnostic gel, discovered that this, too, failed in the same way as the previous digestions had. I decided to try other Kan plasmids
- PCR'd Chris's transcription factor promoter (pTFc)
- Ran nanodrop of pTFc and yesterday's sfGFP and F2620:
| Part # | 230 | 280 | Concentration |
| pTFc A | 1.36 | 1.79 | 196.1 |
| pTFc B | 2.08 | 1.86 | 159.7 |
| sfGFP | 1.89 | 1.87 | 52.9 |
| F2620 | 1.82 | 1.82 | 35.5 |
