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).