Team:TU Darmstadt/Notebook/Methods/PCR with Pfu polymerase

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Revision as of 16:11, 17 October 2014

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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