Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq

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Latest revision as of 11:58, 1 September 2010


PCR with DreamTaq

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

Component Volume Final concentration
DreamTaq Polymerase 10x Buffer (with MgSO4) 5 µl 1x
dNTP 10mM each 5µl 1000µM 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)
Dream Taq Polymerase 0,5µl ~2u/50µl
DMSO 1,25µl
nuclease free water to final volume of 50 µl

!!! DreamTaq Buffer includes Loading Dye !!!

Recommended thermal cycling conditions for DreamTaq Polymerase:

Step Temperature Time Number of Cycles
Initial Denaturation 95°C 3min 1
Denaturation 95°C 0,5min
Annealing 42-65°C (dependent on primer and template) 30sec 25-35
Extension 72°C 1min
Final Extension 72°C 5 min 1
Soak (end) 4°C (on our thermalcyclers 12°C) Indefinite 1

 

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