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

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<b>PCR with Pfu Polymerase</b>
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Source: Promega Usage Information for the Pfu Polymerase
Source: Promega Usage Information for the Pfu Polymerase
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In a sterile, nuclease free PCR-tube mix following components:
In a sterile, nuclease free PCR-tube mix following components:
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Recommended thermal cycling conditions for Pfu Polymerase:
Recommended thermal cycling conditions for Pfu Polymerase:
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|Annealing
|42-65°C (dependent on primer and template)
|42-65°C (dependent on primer and template)
|30sec
|30sec

Latest revision as of 09:43, 17 August 2010


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