From 2010.igem.org
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PCR with DreamTaq
In a sterile, nuclease free PCR-tube mix following components:
| Component
| Volume
| Final concentration
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| DreamTaq Polymerase 10x Buffer (with MgSO4)
| 5 µl
| 1x
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| dNTP 10mM each
| 1µl
| 200µM each
|
| 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
|
| DNA Template
| variable (dependent on DNA conc.)
| <0,5µg/50µl (e.g. 0,3)
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| Dream Taq Polymerase
| 0,5µl
| ~1,25u/50µl
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| DMSO
| 1,25µl
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| nuclease free water to final volume of
| 50 µl
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Recommended thermal cycling conditions for DreamTaq Polymerase:
| Step
| Temperature
| Time
| Number of Cycles
|
| Initial Denaturation
| 95°C
| 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
| 25-35
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| Extension
| 72°C
| 1min
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| Final Extension
| 72°C
| 5 min
| 10
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| Soak (end)
| 4°C (on our thermalcyclers 12°C)
| Indefinite
| 1
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