Team:Ionis Paris/Protocol 8
Realize a mix by adding the following component in a 1,5mL Eppendorf tube. 50µL of mix must be made per desired PCR reaction) Add 50µL of the mix in each 0,2mL PCR tubes. Lid Temperature: 95°C Initial denaturation : 95°C 5 min 25 cycles of : Final extension : 68°C for 5 min Hold at 4°C *precise Tm for each primer Gel purification : Run samples on an electrophoresis gel to check cloning efficiency. Realize a bacterial miniculture of the colony with satisfying PCR results. Inoculate the colony grown on the divided into square petri dish into a 50mL Falcon tube with 5mL LB + appropriate antibiotic (chloramphenicol at 25µg/mL).
Protocol 8 : Colony PCR
Aim: DNA Fragment amplification from bacteria colonies
NB : All components must be vortexed before use
Component Final concentration
Nuclease-free water
10X Standard Taq reaction Buffer 1X
10mM dNTPs 200µM
10µM Forward primer 0.2µM (0.05-1µM)
10µM Reverse primer 0.2µM (0.05-1µM)
Taq polymerase 5 units/50µL PCR
Pick a colony, add a stab of it into a PCR tube and put the rest of the colony on a new annotated and divided into square petri dish
Gently mix and spin down microcentrifuge PCR tubes.
Transfer PCR tubes to a PCR machine with the block preheated to 95°C
Set the following parameters for the PCR reaction :
- 95°C 30 s
- Tm* 1 min
- 68°C; 1 min per kB (amplification)
See the appropriate protocol (Protocol 3 : Electrophoresis )
This will serve for the subsequent miniprep. See the appropriate protocol (Protocol 9 : Miniprep )
