Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.
COMPONENT | VOLUME | FINALCONCENTRATION |
1.autoclaved ultra-filtered water (pH 7.0) | 20.7µL | - |
2.10x PCR Buffer* | 2.5µL | 1x |
3.dNTPs mix (25 mM each nucleotide) | 0.2µL | 200 µM (each nucleotide) |
4.primer mix (25 pmoles/µL each primer) | 0.4µL | 0.4 µM (each primer) |
5.Taq DNA polymerase (native enzyme) | 0.2µL | 1 Unit/25 µL |
6.genomic DNA template (100 ng/µL) | 1.0µL | 100 ng/25 µL |
* The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2).
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