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Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu
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Latest revision as of 09:43, 17 August 2010
Source: Promega Usage Information for the Pfu Polymerase
PCR with 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
