IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/07

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dspB Track

  • cPCR on plates (Streaked)

Colony PCR

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP0.5
sdH2O12.15
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
10uM fw primer (G1004)2
10uM re primer (G1005)2
Total25

PCR Tubes: His, no his, no his A, His A, W

  • 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: 6:46pm
End: 7:49pm

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 1% agarose gel
  • Machine conditions: 0.5x TBE buffer, 100V, 60min

Gel orientation:

Gel orientation
column 1: hiscolumn 5:no-hiscolumn 6: 100bp laddercolumn 8: his Acolumn 9: no his Acolumn 10: W

Results:



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