Realize a mix by adding the following component in a 1,5mL Eppendorf tube. 50µL of mix must be made per desired PCR reaction)
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

Add 50µL of the mix in each 0,2mL PCR tubes.
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 :

Lid Temperature: 95°C

Initial denaturation : 95°C 5 min

25 cycles of :
- 95°C 30 s
- Tm* 1 min
- 68°C; 1 min per kB (amplification)

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.
See the appropriate protocol (Protocol 3 : Electrophoresis )

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).
This will serve for the subsequent miniprep. See the appropriate protocol (Protocol 9 : Miniprep )