Making Electrocompetent Cells
Method
- Grow 5 ml O/N in LB.
- Inoculate 400-500 ml LB with 1/100 volume bacteria from O/N culture.
- Grow to OD600 = 0.6.
- Chill cells on ice for 15 minutes to several hours.
- Pellet cells at 4°C, 2600 rpm, GSA rotor.
- Wash 2X in 200 ml pre-chilled, unused, sterile H2O.
- Pellet as above.
- Resuspend in final volume of 2 ml GYT*.
- Freeze 50-µl aliquots immediately in dry ice/ethanol.
- Store -80°C.
*GYT
- 10% Glycerol
- 0.125% Yeast Extract
- 0.25% Tryptone
- Q with H2O
- Autoclave and store at 4°C
Making Heat Shock Competent Cells
- Inoculate 500 ml LB with 5 ml of a 25-ml O/N culture.
- Grow at 37°C until OD600 = 0.45-0.55
- Chill cells on ice water for 2 hours.
- Pellet cells at 4°C, 2500 g (3700 rpm in GSA rotor) for 15-20 minutes.
- Resuspend cells in 10 to 20 ml ice-cold Ca/Mg buffer**.
- Then bring volume up to 500 ml with the Ca/Mg buffer.
- Incubate on ice for 45 minutes.
- Centrifuge cells 1800 g (3200 rpm GSA rotor) for 10 minutes at 4°C.
- Gently resuspend pellet in 50 ml ice-cold Mg/Cl buffer.
- Pool cells if you have split them up and add 80% glycerol drop by drop, on ice with gentle swirling, to a final concentration of 15% v/v.
- Aliquot cells (0.2-1 ml), freeze on dry ice and store at -80°C.
**Ca/Mg Buffer
- 100mM CaCl2
- 70mM MgCl2
- 40mM Sodium Acetate pH 5.5
- Filter sterilize