Preparation of competent cells

Several super-efficient methods for preparing E. coli competent cells for transformation have been described (e.g. Hanahan's method and Inoue's method). They usually give good results in routine transformation. However, when the protocols are applied to various E. coli strains that are used for genetic studies, quite a few strains tend to give few transformants, probably because those protocols are optimized for specific E. coli strains suitable for transformation, e. g. DH1, DH5, JM109 and their derivatives. The calcium chloride method described below generally gives good results (e. g. 10⁶ transformants/microgram pBR322) for any E. coli strains, although transformation efficiency is relatively lower than the super-efficient methods applied to the optimal strains.

1. Shake E. coli at 37 °C overnight in 3 ml of LB.

2. Add 0.5 ml of the overnight culture into 50 ml of LB in a 200-ml flask. The broth should be prewarmed to 37 °C. Shake the culture at 37 °C.

3. Monitor OD600. When OD600 of 0.35-0.4 is reached, chill the culture on ice. When highest transformation efficiency is not required, I simply harvest cells 100-120 minutes after the inoculation without monitering OD600.

4. Transfer the culture to a sterile centrifuge tube, and collect cells by centrifugation at 6,000 rpm for 8 minutes at 4 °C. Discard the supernatant.

5. Resuspend the cells in 20 ml of ice-cold 50 mM CaCl2. Incubate the resupended cells on ice for 20 minutes. Collect cells by centrifugation as in step 4.

6. Resuspend the cells in 2.5 ml of ice-cold 50 mM CaCl2, or, if you store the competent cells for long period as frozen stock, resuspend the cells in 2.5 ml of ice-cold 50 mM CaCl2 containing 10% glycerol.

7. Use 100-200 microliters of the competent cells for transformation, or dispense the competent cells into aliquotes of 100-200 microliters and freeze in liquid nitrogen for later use.

This method can be easily scaled up and down. When I want to transform an E. coli strain quickly, I inoculate 0.05 ml of an overnight culture into 3 ml of LB, and, after shaking 100-120 min at 37 C, cells are washed with an ice-cold calcium solution. The cells resuspended in 100-150 microliters of the calcium solution are used for transformation.

Transformation

1. Thaw the competent cells on ice if they are stored frozen. Add to 100 microliters of the competent cells appropriate amount of plasmid solutions in TE. The plasmid solution should be less than 5 microliters.

2. Keep the cells on ice for 30 minutes.

3. Quickly transfer the tube to 42 °C water bath. Incubate for 1 minute, then transfer onto ice.

4. Add 1 ml of LB. Incubate at 37 °C for 30 minutes. Spread the cells on agar plate(s) containing appropriate antibiotics.