Troubleshooting for PCR and multiplex PCR

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).