Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion

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{{:Team:LMU-Munich/Templates/Page Header}}  
{{:Team:LMU-Munich/Templates/Page Header}}  
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<b>PCR with Phusion Polymerase</b>
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==PCR with Phusion Polymerase==
Source: Susanne Gebhard
Source: Susanne Gebhard
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|Phusion DNA Polymerase 5x Buffer (with MgSO<sub>4</sub>)
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|Phusion DNA Polymerase 5x Buffer (with 7,5 mmol MgCl<sub>2</sub>)
|10 µl
|10 µl
|1x
|1x
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Recommended thermal cycling conditions for Phusion DNA Polymerase (NOT RIGHT DATA!!!;)
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Recommended thermal cycling conditions for Phusion DNA Polymerase
{|
{|
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|Initial Denaturation
|Initial Denaturation
|98°C
|98°C
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|1-2min
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|1 min
|1
|1
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|Denaturation
|Denaturation
|98°C
|98°C
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|0,5-1min
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|10sec
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|Extension
|Extension
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|72-74°C
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|73°C
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|30sec (2min/kb)
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|30sec (15-30sec/kb)
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|Final Extension
|Final Extension
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|72-74°C
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|73°C
|5 min
|5 min
|1
|1

Latest revision as of 09:58, 31 August 2010


PCR with Phusion Polymerase

Source: Susanne Gebhard

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

Component Volume Final concentration
Phusion DNA Polymerase 5x Buffer (with 7,5 mmol MgCl2) 10 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)
Phusion DNA Polymerase (3u/µl) 0,5µl ~1,25u/50µl
nuclease free water to final volume of 50 µl


Examples for template amounts:

Plasmid DNA: 1-5ng

PCR results: 1-5ng

genomic DNA:


Recommended thermal cycling conditions for Phusion DNA Polymerase

Step Temperature Time Number of Cycles
Initial Denaturation 98°C 1 min 1
Denaturation 98°C 10sec
Annealing 42-65°C (dependent on primer and template) 20sec 25-35
Extension 73°C 30sec (15-30sec/kb)
Final Extension 73°C 5 min 1
Soak (end) 4°C (on our thermalcyclers 12°C) Indefinite 1

 

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