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Team:Lyon-INSA-ENS/Realisation/Protocols/TSS trans
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Revision as of 12:12, 28 July 2011
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 5mL culture of NM522 cells ( or the strain you want to transform ) in LB medium at 37°C. Proceed to the next step when it reaches 0.3-0.4 ( not higher than 0.6 ), which takes approximately 4 hours.
2. Take 1 mL from the previous culture and centrifuge at 6000rpm for 2mn .
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.
11. Plate 200µL on LB medium with the appropriate antibiotic.
