IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/07/13

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dspB Track

Marianne and Vicki

PCR II

  • Dilute PCR product, PCR again, and gel verify
  • Dilution for each sample: 2uL of PCR product + 10uL of H2O = 12uL (1 in 5 dilution)
  • Protocol: common protocols for PCR
Table 1. Microcentrifuge Tubes
123456789101112
  • MMA: 6 diluted samples + 1 extra + 1 water control = 8 samples
  • MMB: 6 diluted samples + 1 extra = 7 samples
Table 2. PCR Tubes
LabelContent
A1
B2
C3
D4
E5
F6
G7
H8
I9
J10
K11
L12
M13
Table 3. Master Mix A
Reagent1X rxn volume (uL)Master Mix
10x rxn buffer2.5x820
10uM FW primer2x816
10uM RE primer2x816
10mM dNTP3x824
sdH2O10.3x892.4
Taq polymerase0.2x81.6
DNA5x840
Total25200
Table 4. Master Mix B
Reagent1X rxn volume (uL)Master Mix
10x rxn buffer2.5x717.5
10uM FW primer2x714
10uM RE primer2x714
10mM dNTP3x721
sdH2O10.3x772.1
Taq polymerase0.2x71.4
DNA5x735
Total25175

PCR Cycles:

  • 95C @ 2 min
  • Cycle 30x:
    • 95C @ 30 sec
    • 65C @ 10 sec
    • 72C @ 80 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1225
End: 1355

Gel verification

  • Protocol: in iGEM training manual
  • Changes: Made 1% agarose gel instead of 0.8% gel

Gel orientation:

Gel orientation
ABCDEF100bp ladderGHIJKLM

Machine conditions: 110V, 45 min, 0.5X TBE Buffer
Results:
[[Image:|300px]]
Vicki Ma 20:37, 13 July 2010 (EDT)



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