Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion


PCR with Phusion Polymerase

Source: Susanne Gebhard

In a sterile, nuclease free PCR-tube mix following components:

Component Volume Final concentration
Phusion DNA Polymerase 5x Buffer (with 7,5 mmol MgCl2) 10 µl 1x
dNTP 10mM each 1µl 200µM each
upstream primer 5-50pmol (from 100pmol/µl e.g. 2,5µl) 0,1- 1 µM
downstream primer 5-50pmol (from 100pmol/µl e.g. 2,5µl) 0,1- 1 µM
DNA Template variable (dependent on DNA conc.) <0,5µg/50µl (e.g. 0,3)
Phusion DNA Polymerase (3u/µl) 0,5µl ~1,25u/50µl
nuclease free water to final volume of 50 µl

Examples for template amounts:

Plasmid DNA: 1-5ng

PCR results: 1-5ng

genomic DNA:

Recommended thermal cycling conditions for Phusion DNA Polymerase

Step Temperature Time Number of Cycles
Initial Denaturation 98°C 1 min 1
Denaturation 98°C 10sec
Annealing 42-65°C (dependent on primer and template) 20sec 25-35
Extension 73°C 30sec (15-30sec/kb)
Final Extension 73°C 5 min 1
Soak (end) 4°C (on our thermalcyclers 12°C) Indefinite 1