Reagent	Amount
Ligated DNA (from step 25)	3 μL
Phusion DNA polymerase buffer 5×	10 μL
10 mM each dNTPs mix	1 μL
2 μM P5 primer (see Table 1)	5 μL
2 μM P7 primer	5 μL
Pushion DNA polymerase	0.5 μL
ddH2O	25.5 μL
Total	50 μL