Competent e coli
- Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent E coli into 5 mL LB broth. Incubate overnight at 37°C with shaking (approximately 225rpm).
- Subculture the overnight culture 1:100 by inoculating 5 mL into 250 mL of LB supplemented with 20mM MgSO4 (0.6 g). Grow the cells in a 1 L flask until the OD600 reaches 0.4-0.6 (usually 5-6 hours, but the time may vary).
- Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C. For a 250 mL culture, use two 250ml centrifuge bottles in a large rotor.
- Gently resuspend the cell pellet in 0.4 original volume of ice-cold TFB1. For a 250ml subculture, use 100ml of TFB1 (50 mL/bottle). Combine the resuspended cells in one bottle. For the remaining steps, keep the cells on ice and chill all pipettes, tubes and flasks.
- Incubate the resuspended cells on ice for 15 minutes.
- Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C.
- Gently resuspend the cells in 1/25 original volume of ice-cold TFB2. For a 250ml subculture, use 10ml of TFB2.
- Incubate the cells on ice for 15-60 minutes, then aliquot 100 µL/tube for storage at -80°C. Quick-freeze the tubes. JM109 competent cells prepared by this method are stable for 1 year.
Note: Competent cells may be conveniently frozen in aliquot of 100 µL cells per tube. I typically transform 50 µL cells with 2-10 µL of a ligation reaction, and you should get between 1x108 to 1x109 cfu's/ug DNA.
LB broth (PSI broth)
For 1 liter:
- 5g Bacto yeast extract
- 20g Bacto Tryptone
- 5g Sodium Chloride (NaCl)
pH 7.6 with potassium hydroxide
- 0.588g potassium acetate
- 2.42g rubidium chloride
- 0.294g calcium chloride
- 2.0g manganese chloride
- 30ml glycerol
pH 5.8 with dilute acetic acid
- 0.21g MOPS
- 1.1g calcium chloride
- 0.121g rubidium chloride
- 15ml glycerol
pH 6.5 with dilute NaOH