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


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