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Team:TU Darmstadt/Notebook/Methods/PCR with Pfu polymerase
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Revision as of 16:09, 16 October 2014
PCR with Pfu polymerase
Short explanation:
PCR (polymerase chain reaction) is a method for exponentially amplifying a fragment of DNA in vitro.
Equipment:
- Micropipettes with sterile tips
- PCR machine
- PCR tubes
Chemicals & consumables: 50 µL batch
- 18 µL Pfu Mastermix 9:25
- 100 ng = 1 µL DNA template
- 1 µL Forward primer (10 µM)
- 1 µL Reverse primer (10 µM)
- up to 50 µL ddH2O
Procedure:
Standard protocol for Pfu Polymerasep>
| # | Temperature | Time | ||
| 1 | 95°C | 00:05:00 | ||
| 2 | 95°C | 00:00:20 | ||
| 3 | 55°C | 00:00:30 | ||
| 4 | 72°C | 2 min/kbp | ||
| 5 | GO TO 2 | REPEAT 30x | ||
| 6 | 72°C | 3 min/kbp | ||
| 7 | 4°C | HOLD |
