Team:TU Darmstadt/Notebook/Methods/Colony PCR with Taq polymerase

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Colony PCR with Taq polymerase

Equipment:

- PCR machine

Chemicals & consumables:

- Sterile Eppendorf Tubes

- LB-agar plate with appropriate antibiotic

- Primers (usually VF2 and VR)

- Sterile pipet tips

Mixtures: 1x Reaction Mixture (25 µL)

- 12,5 µL 2x Taq MM

- 0,5 µL VF2 (10 µM)

- 0,5 µL VR (10 µM)

- 1 µL of colony suspension

- ddH2O to 25 µL

Procedure:

The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.

- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.

- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.

Start the PCR using the following programm and 1X mix.

Run a gel to determine the product length (don't forget the positive control).

#   Temperature   Time
         
1   95°C   00:05:00
         
2   95°C   00:00:30
         
3   55°C   00:00:30
         
4   68°C   1 min/kbp
         
5   GO TO 2   REPEAT 30x
         
6   68°C   1.5 min/kbp
         
7   4°C   HOLD