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Preparation of competent cells:
1. Inoculate a single colony of E. coli cells into 3 ml LB medium. Grow overnight at 37ºC with moderate shaking (250 rpm).
2. Inoculate 4 ml of the culture into 400 ml LB medium in a sterile 2-liter flask. Grow at 37ºC, shaking at 250 rpm, to an OD590 of 0.375.
3. Aliquot culture into eight 50-ml prechilled, sterile polypropylene 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 1600g, 4ºC. (Always allow centrifuge to decelerate without brake for this and all subsequent steps!!!).
5. Pour off supernatant and resuspend each pellet in 10 ml ice-cold CaCl2 solution.
6. Centrifuge cells 5 min. at 1100g, 4ºC. Discard supernatant and resuspend each pellet in 10 ml ice-cold CaCl2 solution. Keep on ice for 30 min.
7. Centrifuge cells 5 min at 1100g, 4ºC. Discard supernatant and resuspend each pellet in 2 ml ice-cold CaCl2 solution.
8. Dispense cells into prechilled, sterile polypropylene tubes (250 μl aliquots are convenient). Freeze immediately at −70ºC.
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Transformation:
1. Thaw on ice one aliquot of frozen competent cells.
2. Mix 2 μl of DNA with 50 μl of cells in a tube on ice. Incubate 30 min.
3. Heat-shock at 42ºC for 45-50 s.
4. Incubate on ice for 2 min.
5. Add 950 μl SOC (or LB) and incubate at 37ºC and 150 rpm for 1-1.5h.
6. Plate onto selective medium.