Competent cell preparation:

1. Plate cells on LB (without antibiotic)

2. Primary inoculation- 37 deg C – 12-16 hours
3. Secondary inoculation- until the O.D reaches 0.5-0.7
4. Keep the cells on ice for 10 mins.
5. Spin at 4000rpm for 10 mins.
6. Resuspend the pellet in 1/4th volume of 100mM MgCl2 and keep in ice for 10 mins.
7. Centrifuge at 4000rpm for 10 mins.
8. Resuspend the pellet in 1/20th volume of 100mM CaCl2 and keep in ice for 20 mins.
9. Centrifuge at 4000rpm for 10 mins.
10. Resuspend in 3mL of (100mM CaCl2 and 10% Glycerol).
11. Aliquot and store at 4C deg for 30 mins and then store in -80deg C.