Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq

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(New page: {{:Team:LMU-Munich/Templates/Page Header}} <b>PCR with DreamTaq</b> In a sterile, nuclease free PCR-tube mix following components: {| |- |Component |Volume |Final concentration |- |Dre...)
 
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{{:Team:LMU-Munich/Templates/Page Header}}  
{{:Team:LMU-Munich/Templates/Page Header}}  
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<b>PCR with DreamTaq</b>
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==PCR with DreamTaq==
In a sterile, nuclease free PCR-tube mix following components:
In a sterile, nuclease free PCR-tube mix following components:
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|-
|-
|dNTP 10mM each
|dNTP 10mM each
-
|1µl
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|5µl
-
|200µM each
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|1000µM each
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|Dream Taq Polymerase (3u/µl)
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|Dream Taq Polymerase
|0,5µl
|0,5µl
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|~1,25u/50µl
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|~2u/50µl
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|-
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!!! DreamTaq Buffer includes Loading Dye !!!
Recommended thermal cycling conditions for DreamTaq Polymerase:
Recommended thermal cycling conditions for DreamTaq Polymerase:
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|72°C
|72°C
|5 min
|5 min
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|10
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|1
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Latest revision as of 11:58, 1 September 2010


PCR with DreamTaq

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

Component Volume Final concentration
DreamTaq Polymerase 10x Buffer (with MgSO4) 5 µl 1x
dNTP 10mM each 5µl 1000µM 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)
Dream Taq Polymerase 0,5µl ~2u/50µl
DMSO 1,25µl
nuclease free water to final volume of 50 µl

!!! DreamTaq Buffer includes Loading Dye !!!

Recommended thermal cycling conditions for DreamTaq Polymerase:

Step Temperature Time Number of Cycles
Initial Denaturation 95°C 3min 1
Denaturation 95°C 0,5min
Annealing 42-65°C (dependent on primer and template) 30sec 25-35
Extension 72°C 1min
Final Extension 72°C 5 min 1
Soak (end) 4°C (on our thermalcyclers 12°C) Indefinite 1

 

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