Matt:LabNotes/2012-5-9

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qPCR with DNA standards[edit]

DNA Standards

  1. 10 ng/ul
  2. 1 ng/ul
  3. 0.1 ng/ul
  4. 0.01 ng/ul

Procedure: Standard qPCR

  • 1 ul DNA template
  • 15 ul 2x KAPA MasterMix
  • 0.6 ul Forward primer
  • 0.6 ul Reverse primer
  • 12.8 ul H2O

Cycles of: 95C 0:10 58C 0:30 72C 2:00

Removed 2 tubes from each of the 4 samples every time one of their curves began to increase, suggesting that it reached levels differentiable from background noise. The DNA concentrations were measured

Results[edit]

Measured DNA concentrations

Cycles 10 ng/µl 1 ng/µl 0.1 ng/µl 0.01 ng/µl
0 10 1 0.1 0.01
4 24.2 2.52 0.24 0.0479
6 78.1 9.5 0.735 0.0953
10 162.9 79.2 8.31 1.07
14 72.3 10.2

Conclusions

  • Threshold concentration of dsDNA for detection with SYBR Green is approx 10 ng/ul
  • Very low concentrations of template DNA amplify just as efficiently if not more, even though real time fluorescence not detectable
  • First 4 cycles are not as efficient as ideal exponential growth predicts