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From 2011.igem.org

Materials:

"overnight" cell culture

0.1M CaCl2 solution

0.1M CaCl2 15%glycerol solution

Procedure:

1. Grow an "overnight" cell culture of the strain you want to turn competent.

2. Make a 1:100 dilution of the "overnight" cell culture and grow to a OD600 of 0.3-0.5(we inoculate 1ml of overnight into 100 ml of LB)

3. split the cell culture into two 50ml centrifuge tubes.

4. centrifuge at 4000rpm at 4°C for 10 min.(ALWAYS KEEP THE CELLS ON ICE FROM THIS POINT ONWARD, THIS IS VERY IMPORTANT)

5. discard the supernatant.

6. GENTLY resuspend the cells using 20ml of cold 0.1M CaCl2 solution for each centriuge tube.

7. incubate on ice for 30 min.

8. centrifuge at 4000rpm at 4°C for 10 min.

9. discard supernatant.

10. resuspen each tube using 3ml of cold CaCl2 0.1M 15% glycerol solution.

11. aliquot however you seem fit in 2ml microtubes or cryovials.

12. store at -80 as soon as you are done.

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