Team:Lyon-INSA-ENS/Realisation/Protocols/CaCl2 trans

CaCl2 chemical transformation

This protocol aims at inserting plasmids into NM522 strains

Procedure

1. Monitor the OD600 of a 50mL culture of NM522 cells in LB medium at 37°C. Proceed to the next step when it reaches 0.2-0.4 ( not higher than 0.5 ), which takes approximately 2-3 hours.

2. Aliquot the culture into 10mL fractions.

3. Incubate on ice for 15mn

4. Centrifuge at 4°C, 10mn, 5000rpm

5. Empty the supernageant

6. Add 5mL cold and sterile CaCl2 100mM

7. Incubate 20mn on ice

8. Centrifuge at 4°C, 5mn, 5000rpm

9. Empty the supernageant and resuspend ( do not vortex ) in 1mL CaCl2

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