IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/13

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dspB Track

Plate results:

  • 50uL HR - no colonies
  • 450uL HR - ~20 colonies (most of them red); (A, D - white?; B, C - pink?)
  • 50uL HC - no colonies
  • 450uL NC - no colonies
  • 50uL NR - 3 colonies (2red) (A)
  • 450uL NR - ~20 colonies (~ half of them red) (A, B, C, D)
  • 50uL NC - colonies
  • 450uL NC - 34 colonies (A, B, C, D)

Colony PCR

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1680
10mM dNTP0.5x168
sdH2O12.15x16194.4
Phusion polymerase0.1x161.6
MgCl22x1632
DMSO - 5%1.25x1620
10uM fw primer (G1004)2x1632
10uM re primer (G1005)2x1632
Total25400
  • Pick colony from respective plate and place in respective tubes

PCR Cycles:

  • 98C @ 3min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1:53pm
End: 2:57pm

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 0.6g agarose/70mL 0.5x TBE buffer
  • Machine conditions: 0.5x TBE buffer, 100V, 50min

Gel orientation:

Gel orientation
450uL NR A450uL NR B450uL NR C450uL NR D50uL NR A450uL NC A450uL NC B100 bp ladder450uL NC C450uL NC D450uL HR A450uL HR B450uL HR C450uL HR DW

Results:
File:100913 dspB 28.tif

Re-Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 0.6g agarose/70mL 0.5x TBE buffer
  • Machine conditions: 0.5x TBE buffer, 100V, 50min

Gel orientation:

Gel orientation
450uL NC A450uL NC B450uL NC C450uL NC D50uL HR A450uL HR B450uL HR C100 bp ladder450uL HR D C450uL NR A450uL NR B450uL NR C450uL NR D50uL NR AW1kb ladder

Results:
File:100913 dspB 29.tif

Began O/N Culture

  • in 37C shaking incubator at 11:30pm