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Protocols

Preparation of competent Bacterial cells

1- Preparation of the Bacterial culture

Recuperate the overnight bacterial culture
Determine the OD600 in a 1 ml Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
Put the Erlenmeyer in an incubator for about an hour at 37˚C.
test the OD600 for the new culture
the OD600 should be between 0.4 and 0.6

All the handling done in a 15cm radius of an open flame for optimal sterility

2- Preparation of Buffer Tbf1 and Tbf2

Tbf1 buffer	Total volume 80mL
KAc 1M	2.4 mL
MnCl2 0.5M	8 mL
KCl 1M	8 mL
CaCl₂ 0.1M	8 mL
GlY 80%	15mL
H₂O	38.6 mL

Tbf2 buffer	Total volume 8 mL
NaMOPS 0.2 M	400 µL
CaCl₂ 0.1 M	6 mL
Gly 80%	1.5 mL
KCl 1 M	80 µL
H₂O	500 µL

3- Preparation of Competent Bacterial cells

Transfer the Bacterial culture in 50 ml Falcon tubes
Centrifuge for 10 minutes at 3500 rpm in cold
Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for 50 ml of bacteria
Centrifuge for 5 minutes at 3500 rpm in cold
Poll in all bacterial culture in a single tube
Remove the supernatant then re-suspend the pellet in 8 ml of Tbf2 for 200 ml of bacteria
Allocate 220 µl of competent Bacterial cells in each eppendorf tube
instant freeze the eppendorf tubes in liquid nitrogen
Conserve the tubes at -80˚C

NOTE: all handling done in a cold room. To re-suspend the pellet a hard jerking action applied on the tube. Alternatively, put on wheel for 10 minute.

Team:Aix-Marseille/Experiments/Protocols