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Revision as of 10:43, 29 September 2010
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PCR with Pfu Polymerase
Source: Promega Usage Information for the Pfu Polymerase
In a sterile, nuclease free PCR-tube mix following components for 50µl:
| Component
| Volume/Final concentration
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| Taq DNA 10x Buffer (without MgCl2)
| 5 µl
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| dNTP 0.2mM each
| 5µl
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| upstream primer
| 0,1- 1 µM
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| downstream primer
| 0,1- 1 µM
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| DNA Template
| 10pg-1µg
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| Taq DNA Polymeras(3u/µl)
| 1,25u
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| nuclease free water to final volume of
| 50 µl
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Recommended thermal cycling conditions for Taq Polymerase:
| Step
| Temperature
| Time
| Number of Cycles
|
| Initial Denaturation
| 95°C
| 1-3min
| 1
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| Denaturation
| 95°C
| 0,5min
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| Annealing
| 42-65°C (dependent on primer and template)
| 30sec
| 25-40
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| Extension
| 72°C
| 1min (1min/kb)
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| Final Extension
| 72°C
| 5-15 min
| 1
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| Soak (end)
| 4°C (on our thermalcyclers 12°C)
| Indefinite
| 1
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