Team:University College London/Protocols/Phusion

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