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Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq
From 2010.igem.org
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{{:Team:LMU-Munich/Templates/Page Header}} | {{:Team:LMU-Munich/Templates/Page Header}} | ||
| - | + | ==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 | ||
| - | | | + | |5µl |
| - | | | + | |1000µM each |
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|Dream Taq Polymerase | |Dream Taq Polymerase | ||
|0,5µl | |0,5µl | ||
| - | |~ | + | |~2u/50µl |
|- | |- | ||
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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 | ||
| - | | | + | |1 |
|- | |- | ||
Latest revision as of 11:58, 1 September 2010
In a sterile, nuclease free PCR-tube mix following components:
!!! DreamTaq Buffer includes Loading Dye !!!
Recommended thermal cycling conditions for DreamTaq Polymerase:
PCR with DreamTaq
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
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
