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Revision as of 20:57, 4 August 2014

Transformation of competent cells by electroporation

1. Cells preparation:

With the precultures made the day before, add 200µl of bacteria in 15ml of LB medium and add the appropriate antibiotic in a Falcon tube.

Shake vigorously at 30°C until the OD600nm reaches 0,6.

Then, place in an ice bath for 10 minutes and centrifuge the cells for 10 min at 4,000 rpm at 4 °C.

Resuspend the cell pellet in the same initial volume of Glycerol 10% cold and sterile. Centrifuge 5 minutes at 4000rpm at 4°C.

Then, centrifuge the cells for 10 min at 4,000 rpm at 4 °C, and resuspend the cell pellet in the same initial volume of Glycerol 10% cold and sterile. Centrifuge 5 minutes at 4000rpm at 4°C

Then, centrifuge the cells for 10 min at 4,000 rpm at 4 °C.

Resuspend the cell pellet in 100µl of Glycerol 10% cold and sterile.

2. Cells transformation:

In 2 electroporation tubes, put 50µl of your bacterial culture in each tube and add Xµl of your plasmid in one! The tube without plasmid will be the negative control.

Proceed to the electroporation, verify your variable (close to 6)

Add immediately after the electroporation 1ml of LB medium cold!

Leave 1 to 2 hours at 37°C.

Then, spread on dishes at different concentrations: 100µl, 200µl and 150µl concentrate. Put just 100µl for the negative control dish.