Initial concentration	Volume per 20 μl reaction	Final amount per 20 μl reaction
Phusion HF buffer (5 ×)	4 μl	1 ×
dNTP mix (40 mM)	0.4 μl	0.8 mM
Phusion high-Fidelity DNA polymerase	0.25 μl	0.5 U
Vector DNA	–	100 ng
Insert DNA	Variable	0, 65.4, 130.8 and 196.14 ng (for V:I molar ratio 1:0, 1:1, 1:2, and 1:3)
Nuclease-free water	Upto 20 μl	–