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

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Examples for template amounts: Plasmid DNA: 1-5ng
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                              PCR results: 1-5ng
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                              genomic DNA:

Revision as of 08:09, 31 August 2010


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 MgSO4) 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 (NOT RIGHT DATA!!!;)

Step Temperature Time Number of Cycles
Initial Denaturation 98°C 1-2min 1
Denaturation 98°C 0,5-1min
Annealing 42-65°C (dependent on primer and template) 20sec 25-35
Extension 72-74°C 30sec (2min/kb)
Final Extension 72-74°C 5 min 1
Soak (end) 4°C (on our thermalcyclers 12°C) Indefinite 1