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From 2012.igem.org

Phusion High Fidelity Polymerase

Component	20 µl Reaction	50 µl Reaction	Final Concentration
Nuclease-free water	to 20 µl	to 50 µl
5X Phusion HF or GC Buffer	4 µl	10 µl	1X
10 mM dNTPs	0.4 µl	1 µl	200 µM
10 µM Forward Primer	1 µl	2.5 µl	0.5 µM
10 µM Reverse Primer	1 µl	2.5 µl	0.5 µM
Template DNA	variable	variable	< 250 ng
DMSO (optional)	(0.6 µl)	(1.5 µl)	3%
Phusion DNA Polymerase	0.2 µl	0.5 µl	1.0 units/50 µl PCR

Thermocycling conditions for a routine PCR: Initial denaturation: 98°C 30 seconds

25–35 cycles: 98°C 5–10 seconds 45–72°C 10–30 seconds 72°C 15–30 seconds/kb

Final extension: 72°C 5–10 minutes

Hold: 4°C