Stock PCR primer solution preperation: 100 uM (20 nmol in 200 ul RNase-free water); working solution: 10 uM.
Recipe for 25 ul reaction volume
DNA---------------------------25 ng
10x PCR buffer-----------------2.5 ul
50mM MgCl2------------------1.25 ul
5 mM dNTPs-------------------2.5 ul
10 uM Forward primer----------1.0 ul
10 uM Reverse primer-----------1.0 ul
5 units/ul Taq polymerase--------0.2 ul
Add ddH2O to ----------------25 ul
PCR reaction:
1. Denature DNA at 95°C for 7 minutes
2. PCR cycles:
94°C-------30 sec
55°C-------20 sec
72°C-------45 sec
for 35 cycles
