IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/23

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dspB Track

Restriction Digest Chlor backbone psb1C3

  • Protocol: biobrick (digesting)
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL
Buffer 25
BSA0.5
ddH2O37
Total42.5
  • Add: 5uL of backbone
  • Enzymes: EcoRI and SpeI (1uL each to each tube)

Transformation

  • Transform ligation mixes from Aug 13 2010
  • Tubes (ligation mixes): 1,2,3,4,5,6,7,8,11,12,13,14,15,AA,AB,TA,TB,CA
  • Protocol: Common protocols - SOP
    • Changes: Add 10uL ligation mixes instead of 1uL

Note: 14,15, AA,AB,TA,TB,CA were transformed with 2uL of ligation mix

  • Alina ligated His (7) and No-his (23). Will also transform: also add 2uL of his + no-his ligation mixes
  • Transformants put in 37C incubator at 1450, taken out at 1617

Control Experiment

  • RD Chlor (psb1C3) and Amp (psb1A3) @ EcoRI, XbaI, PstI cut sites
  • Protocol: RD Protocol (biobrick)
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL
Buffer 25
BSA0.5
ddH2O37
Total42.5
  • Add DNA: 5uL of backbone
  • Enzymes: EcoRI, XbaI, PstI (1 uL each to each tube)


QS Track

  • Like the dspB track, a bacterial mat grew when transformants were spread on chloramphenicol plates.
  • A bacterial mat also grew when transformants were spread on LB + 20 μL chloramphenicol (1000X).
  • This suggests a possible problem with the chloramphenicol aliquots (1000X) used.
  • Marianne remade the chloramphenicol aliquots using 3 different sources of chloramphenicol.
  • Phillip made chloramphenicol plates (20) using Marianne's C3 aliquots (chlor source: last year's parafilm sealed bottle)
  • From yesterday's leftover ligation mixtures, 5 μL was transformed again by Vicki (following UBC iGEM procedures).
  • Also started an overnight culture of pcN33. Host is Staph aureus RN4220. Used LB broth (no erm in lab).
  • Made an antibiotic test of possible problematic chloramphenicol aliquots mentioned earlier. 10 μL of chloramphenicol aliquot was added to 10 mL LB. A stab of stock DH5(alpha)was inoculated in this medium. Expect no growth.

Biofilm Track

Melody

Biofilm Growth Protocol Day 1 (100823M+E)

  • Performed Day 1 of the Biolfilm Protocol
  • O/N cultures of Control, RN4220 and 8325-4 in 5mL of TSB
  • Into incubator @ 1425