From 2010.igem.org
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PCR with Phusion Polymerase
Source: Susanne Gebhard
In a sterile, nuclease free PCR-tube mix following components:
| Component
| Volume
| Final concentration
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| Phusion DNA Polymerase 5x Buffer (with MgSO4)
| 10 µl
| 1x
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| dNTP 10mM each
| 1µl
| 200µM each
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| upstream primer
| 5-50pmol (from 100pmol/µl e.g. 2,5µl)
| 0,1- 1 µM
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| downstream primer
| 5-50pmol (from 100pmol/µl e.g. 2,5µl)
| 0,1- 1 µM
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| DNA Template
| variable (dependent on DNA conc.)
| <0,5µg/50µl (e.g. 0,3)
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| Phusion DNA Polymerase (3u/µl)
| 0,5µl
| ~1,25u/50µl
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| nuclease free water to final volume of
| 50 µl
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Examples for template amounts: Plasmid DNA: 1-5ng
PCR results: 1-5ng
genomic DNA:
Recommended thermal cycling conditions for Phusion DNA Polymerase (NOT RIGHT DATA!!!;)
| Step
| Temperature
| Time
| Number of Cycles
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| Initial Denaturation
| 98°C
| 1-2min
| 1
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| Denaturation
| 98°C
| 0,5-1min
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| Annealing
| 42-65°C (dependent on primer and template)
| 20sec
| 25-35
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| Extension
| 72-74°C
| 30sec (2min/kb)
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| Final Extension
| 72-74°C
| 5 min
| 1
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| Soak (end)
| 4°C (on our thermalcyclers 12°C)
| Indefinite
| 1
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