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

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(New page: {{:Team:LMU-Munich/Templates/Page Header}} <b>PCR with Taq Mastermix</b> Source: Promega Usage Information for the PCR Master Mix (containing Taq Plymerase) PCR Master Mix has been opti...)
 
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<b>PCR with Taq Mastermix</b>
<b>PCR with Taq Mastermix</b>
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Source: Promega Usage Information for the PCR Master Mix (containing Taq Plymerase)
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Source: Promega Usage Information for the PCR Master Mix (containing Taq Polymerase)
PCR Master Mix has been optimized for use in routine PCR reactions for amplifying DNA template in the range of 0.2-0kb
PCR Master Mix has been optimized for use in routine PCR reactions for amplifying DNA template in the range of 0.2-0kb
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Protocol:
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.
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.
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In a sterile, nuclease free PCR-tube mix following components for a 100µl reaction volume:
In a sterile, nuclease free PCR-tube mix following components for a 100µl reaction volume:
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::final: 5'
::final: 5'
:D. Refrigeration
:D. Refrigeration
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::4°C actually; our thermo cycler can just go down to 12°C
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::4°C (our thermo cycler can just go down to 12°C)
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:Cycle Number
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:E.Cycle Number
::25-30 cycles
::25-30 cycles
{{:Team:LMU-Munich/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 09:53, 21 September 2010


PCR with Taq Mastermix


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

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 (our thermo cycler can just go down to 12°C)
E.Cycle Number
25-30 cycles

 

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