Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix

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Revision as of 09:52, 21 September 2010


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PCR with Taq Mastermix

Source: Promega Usage Information for the PCR Master Mix (containing Taq Plymerase)

PCR Master Mix has been optimized for use in routine PCR reactions for amplifying DNA template in the range of 0.2-0kb

Protocol:

Thaw the PCR Master Mix at room temperature. Vortex the Master Mix and then spin it briefly in a microcentrifuge to collect the material in the bottom of the tube.

In a sterile, nuclease free PCR-tube mix following components for a 100µl reaction volume:

Component	Volume	Final concentration
PCR Master Mix, 2X	50 µl	1x
upstream primer, 10µM (Bio seq f)	1.0-10.0µl	0,1- 1 µM
downstream primer, 10µM (Bio seq r)	1.0-10.0µl	0,1- 1 µM
DNA Template	1-5µl	<250ng
nuclease free water to final volume of	100 µl

Guidelines for Amplification by PCR

A. Denaturation:

initial: 95°C 2'

subsequent: 30 - 1'

B. Annealing:

53°C 30 - 1'

C. Extension

72°C 1'/kb of DNA to be amplified

final: 5'

D. Refrigeration

4°C actually; our thermo cycler can just go down to 12°C

E.Cycle Number

25-30 cycles

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 <b>PCR with Taq Mastermix</b>
 Source: Promega Usage Information for the PCR Master Mix (containing Taq Plymerase)