Matt:LabNotes/2012-5-9
Jump to navigation
Jump to search
qPCR with DNA standards[edit]
DNA Standards
- 10 ng/ul
- 1 ng/ul
- 0.1 ng/ul
- 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