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

CaCl2 chemical transformation

This protocol aims at inserting plasmids notably into NM522 strains, or any other E.Coli strain

It has been found more efficient than the CaCl2 transformation protocol, thus we do not recommend using it.

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 0.6 ), which takes approximately 4 hours.

2. Take x mL from the previous culture and centrifuge at .

3. Resuspend into 100µL of TSS. Starting from this step, keep the bacteria on ice.

4. Incubate on ice for 5-10mn.

5. Add 5µL of the DNA to be transformed.

6. Incubate on ice for 10mn.

7. Heat shock the bacteria by heating them at 42°C for 50s.

8. Incubate on ice for 2mn.

9. Add 900µL LB and mix by inverting the tube.

10. Incubate for 1h at 37°C.