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