From 2010.igem.org
|
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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