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Escherichia coli cells are grown to log phase. Cells are concentrated by centrifugation and resuspended in a solution containing calcium chloride. Exposure to calcium ions enables the cells to take up DNA, or to become competent. Plasmid DNA is mixed with the cells and presumably adheres to them. The mixture of DNA and cells is then heat shocked, which allows the DNA to efficiently enter the cells. The cells are grown in nonselective medium to allow synthesis of plasmid-encoded antibiotic resistance proteins, then plated on antibiotic-containing medium to allow selection of plasmid-containing colonies.

NOTE: All materials coming into contact with E. coli must be sterile.

Preparing Competent Cells

1. Inoculate a single colony of E. coli cells into 50 ml LB medium. Grow overnight at 37° C with moderate shaking (250 rpm).
2. Inoculate 2 ml of the culture into 200 ml LB medium in a sterile 2 liter flask. Grow at 37° C, shaking (250 rpm), to an OD590 of 0.375. This procedure requires that cells be growing rapidly (early- or mid-log phase). Accordingly, it is very important that the growing cells have sufficient air. A 1-liter baffle flask can be used instead of the 2 liter flask. Overgrowth of culture (beyond OD590 of 0.4) decreases the efficiency of transformation.
3. Aliquot culture into 8-25 ml prechilled, sterile corex tubes and leave the tubes on ice 5 to 10 min.
Cells should be kept cold for all subsequent steps.
4. Centrifuge cells 7 min at 3000 rpm, 4 C, in a JA-20 rotor; decelerate with 1/2 brake.
5. Pour off supernatant and resuspend each pellet in 5 ml of ice cold CaCl2 solution. Resuspension should be performed very gently and all cells kept on ice.
6. Centrifuge cells 5 minutes at 2500 rpm, 4 C. Discard supernatant and resuspend each pellet in 5 ml of cold CaCl2 solution. Keep resuspended cells on ice for 30 minutes.
7. Pellet cells at 4 C, 2500 rpm for 5 minutes. Resuspend each pellet in 2 ml of ice cold CaCl2 solution. It is important to resuspend this final pellet well. The suspension may be left on ice for several days. For many strains (e.g. DH1) competency increases with increasing time on ice, and reaches a maximum at 12 to 24 hours.
8. Dispense cells into prechilled, sterile polypropylene tubes (100 µl aliquots are convenient). Freeze immediately at -70° C.

Assessing Competency of Cells

9. Use 10 ng of pBR322 to transform 100 µl of competent cells according to the steps provided below. Plate appropriate aliquots (1, 10, and 25 µl) of the transformation culture on LB/ampicillin plates and incubate at 37° C overnight.
10. The number of transformant colonies per aliquot volume (µl) x 10^5 is equal to the number of transformants per microgram of DNA.Transformation efficiencies of 10^6 to 10^8 are obtained. Competency of strains decreases very slowly over months of storage time.