Appendix A: Competent Cells Procedure

1. Pick a single colony from a stock plate of the desired strain and grow overnight in 5mL of LB broth at 37°C with vigorous shaking.
2. Inoculate 1.0mL of the overnight culture into 60mL LB in a sterile Erlenmyer flask and grow at 37°C to log phase (OD600 = 0.4-0.5).
3. Pellet the cells in two cold, sterile oak ridge tubes (25 mL culture per tube) at 4°C 5000rpm for 5-10 minutes.
4. Decant the supernatant and resuspend each pellet in 10mL cold, sterile 0.1mol/L MgCl2 with 5mmol/L Tris-HCl. Gently resuspend the cells and hold on ice for 20-30 minutes.
5. Pellet the cells as in step 3.
6. Decant the supernatant and resuspend the cells in 5mL of cold, sterile TG salts.
7. Pellet the cells as before.
8. Decant the supernatant and resuspend the cells in 1mL of cold, sterile TG salts. Hold on ice in a cold room for 4-24 hours.
9. Dispense 200µL aliquots of competent cells into prechilled 500µL microfuge tubes and freeze at –80°C.

Note: keep the cells on ice at all times except when in the 4°C centrifuge. Don’t hold the cells in the TG salts longer than 24 hours without freezing as cell viability decreases. TG salts: 75mmol/L CaCl2, 6mmol/L MgCl2 5mmol/L Tris-HCl and 15% glycerol. Autoclave.