Team:Ionis Paris/Protocols 8

Protocol 9 : Colony PCR

Aim: DNA Fragment amplification from bacteria colonies

Liquid LB medium preparation (1L)

Realize a mix by adding the following component in a 1,5 mL tubes (realize 20 µL of mix per desired PCR reaction)
NB : All components must be vortexed before use

Add 20 µL of the mix in each 0,2 mL PCR tubes.
Pick a colony and add a stab of it into a PCR tube.
Gently mix and spin down microcentrifuge. Transfer PCR tubes to a PCR machine with the block preheated to 95°C
Set the following parameters for the PCR reaction :

  • Lid Temperature: 95°C

  • Initial denaturation : 95°C 30 s

  • 25 cycles of :
    - 95°C 30s
    - 58°C 60s
    - 68°C; 1 min per kB (amplification)

  • Final extension : 68°C for 5 min

  • Hold at 4°C