Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu

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Latest revision as of 09:43, 17 August 2010


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PCR with Pfu Polymerase

Source: Promega Usage Information for the Pfu Polymerase

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

Component	Volume	Final concentration
Pfu DNA Polymerase 10x Buffer (with MgSO₄)	5 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)
Pfu DNA Polymerase (3u/µl)	0,5µl	~1,25u/50µl
nuclease free water to final volume of	50 µl

Recommended thermal cycling conditions for Pfu Polymerase:

Step	Temperature	Time	Number of Cycles
Initial Denaturation	95°C	1-2min	1
Denaturation	95°C	0,5-1min
Annealing	42-65°C (dependent on primer and template)	30sec	25-35
Extension	72-74°C	2-4min (2min/kb)
Final Extension	72-74°C	5 min	1
Soak (end)	4°C (on our thermalcyclers 12°C)	Indefinite	1

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 Source: Promega Usage Information for the Pfu Polymerase
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