Transformation of DNA

1. Remove a trace of E. coli cells from the glycerol stock vial with a sterile toothpick or inoculating loop, and streak it out on LB-agar plates containing an appropriate concentration of the relevant selective antibiotic(s) (see Antibiotics). If the host strain has already been cultured and stored at 2–8°C (cultures can be stored at 2–8°C for up to 3 months without any significant loss of viability), streak out bacteria from those stocks.
2. Incubate at 37°C overnight.
3. Pick a single colony and inoculate 10 ml LB medium containing relevant antibiotic(s). Grow overnight at 37°C.
4. Add 1 ml overnight culture to 100 ml prewarmed LB medium containing the relevant antibiotic(s) in a 500 ml flask, and shake at 37°C until an OD₆₀₀ of 0.5 is reached (approximately 90–120 min).
5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.
6. Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4°C).
7. Discard the supernatant carefully. Always keep the cells on ice.
8. Resuspend the cells gently in cold (4°C) TFB1 buffer (30 ml for a 100 ml culture) and keep the suspension on ice for an additional 90 min.
9. Collect the cells by centrifugation (5 min, 4000 x g, 4°C).
10. Discard the supernatant carefully. Always keep the cells on ice.
11. Resuspend the cells carefully in 4 ml ice-cold TFB2 buffer.
12. Prepare aliquots of 100–200 µl in sterile microcentrifuge tubes and freeze in liquid nitrogen or a dry-ice–ethanol mix. Store the competent cells at –70°C.