Preparation of competent cells not to be frozen

1. From a fresh master plate inoculate 2 cultures at 2.5mLs each.
2. Next morning dilute the saturated culture 1:200 into fresh LB. Make 2 25 mL cultures.
3. Allow the cultures to grow to an OD₆₀₀ of ~0.5. This should take approximately 2 hours, depending on the cell line. If the OD₆₀₀ reaches >0.7 discard the cultures.
4. Transfer the cultures to sterile 50 mL centrifuge tubes. Allow the cells to cool on ice for 10 minutes.
5. Pellet the cells in the refrigerated 4 C centrifuge at 2800 RPM for approximately 10 minutes.
6. Pour off the supernatant and re-suspend each cell pellet in 10mL of ice cold, sterile CaCl2.
7. Incubate the cells on ice for 10 minutes.
8. Pellet the cells again in the refrigerated 4 C centrifuge at 2800 RPM for approximately 10 minutes.
9. Pour off the supernatant. Re-suspend cells in a total of 1mL of ice cold 1mL of ice cold 100mM CaCl2.
10. Allow cells to rest on ice for a minimum of 1 hour before use. Cells can be stored at 4 C for up to 48 hours and remain competent.