Team:Stanford/Notebook/Lab Work/Week 6

Spring: Brainstorming | Spring Meetings

Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries

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)

• 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:

8/4 Wednesday

8/5 Thursday

8/6 Friday