Troubleshooting for PCR and multiplex PCR
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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 |
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|
2.10x PCR Buffeundefined |
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 |
