Team:Stanford/Notebook/Lab Work/Week 6

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Contents

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


8/4 Wednesday

8/5 Thursday

8/6 Friday