Component	Amount (μl)	Final concentration
Herculase II PCR buffer, 5×	10	1×
dNTP, 100 mM (25 mM each)	1	2 mM
Out-Fwd or Out-Rev primer, 10 μM	1	0.2 μM
In-Fwd or In-Rev primer, 10 μM	1	0.2 μM
Herculase II fusion polymerase	1
MgCl2, 25 mM	2	1 mM
DNA template	1	2 ng μl−1
ddH2O	33
Total	50