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Overview
Materials
- 96-well PCR plate
- AmpliTaq DNA Polymerase 5 U/µl (Roche)
- AmpliTaq Buffer 10 x
- dNTP mix 10mM each nucleotide
- ddH2O
- primers:
Procedure
I PCR reaction
| 11µl single reaction | 60xMaster | 115xMaster |
| H2O | 7.5µl | 450 | 862 |
| 10x Amplitaq buffer | 1.1µl | 66 | 126.5 |
| 10mM dNTP | 0.22µl | 13.2 | 25.3 |
|
| VF2 primer 10 µM | 0.55µl | 33 | 63.3 |
| VR primer 10 µM | 0.55µl | 33 | 63/3 |
| AmpliTaq 5U/µl | 0.1µl | 6 | 11.5 |
|
| template DNA | 1µl | -- | -- |
|
| PCR Program |
|---|
| 10' @95°C |
| 30x | (30" @95°C; 15" @64; text@72°C); |
| optional: 10' @72°C |
| ∞ 4°C |
- extension time text = (kb insert length) × 1' (1 min)
|
II Agarose Gel
Notes
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raik:
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References
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