Team:TU Darmstadt/Protocols/Colony PCR

Colony PCR

About

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.

Materials

• Sterile Eppendorf Tubes
• LB-agar plate with appropriate antibiotic
• Primers (usually VF2 and VR)
• PCR machine
• Sterile pipet tips

Procedure

1. Pick one colony with a sterile tip and suspend in 10 µL of DI H₂O
2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
3. Start the PCR using the following programm and 1X mix
4. Run a gel to determine the product length (don't forget the positiv control)

Reaction mixture

1X reaction mix contains:

• • 2 µL of 10x Thermopol Reaction Buffer
  • 0,4 µL of dNTPs (10 mM each)
  • 0,3 µL of Taq DNA Polymerase
  • VF2 (10 pmol)
  • VR (10 pmol)
  • 0,6 µL of DMSO
  • 1 µL of colony suspension
  • DI water to 20 µL

• PCR program

#	Temperature	Time
1	95 °C	00:05:00
2	95 °C	00:00:20
3	62 °C	00:00:30
4	68 °C	00:02:00
5	GO TO 2	REPEAT 30x
6	68 °C	00:05:00
7	4 °C	HOLD

Elongation time (step 4 depends on the length of the fragment 1 min per kb)).