Protocol
Protocol used for the Lab Job of making competent cells.
Materials
Prepare following solutions:
TFB1 Concentration FW
Rubidium Chloride 100 mM 120.92
Manganese Chloride (MnCl2 H20) 50 mM 197.91
Potassium Acetate 30 mM 98.14
Calcium Chloride (CaCl2H2O) 10 mM 147.02
Glycerol 15%*
Adjust to pH 5.8 with dilute (0.2%; 1.0 M) acetic acid. Do NOT overshoot. Filter sterilize. Store at room temp but bring to 4C before use.
TFB2 Concentration FW
MOPS 10 mM 209.3
Rubidium Chloride 10 mM 120.92
Calcium Chloride 75 mM 147.02
Glycerol 15%
Adjust pH to 6.5 with KOH. Do NOT overshoot. Filter sterilize. Store at room temp but bring to 4C before use.
- 2X YT media
- 16 g Bacto tryptone
- 10 g Bacto yeast extract
- 5g NaCl
Add 900 mL water and adjust to pH 7.0 with 5N NaOH.
Bring up to 1000L and autoclave.
Or TEKZR097 in CCF 48h delivery (~$40)
Methods
Step 1: Culture bacteria.
- Streak/plate bacteria of choice on LB agar plate.
- Inoculate single colony into starter culture of 20 mL SOC media in 125mL Erlenmeyer flask.
- Incubate overnight in 30C or 37C shaker.
- Inoculate growth culture 1:100 with starter culture. (2.5 mL of starter into 250 mL 2X YT media in 1L Erlenmeyer flask). Put in 37C shaker.
- Grow until OD600 reaches 0.4 to 0.6 (~5h)
- After this point, keep everything cold. Work in cold room and pre-chill all supplies.
Step 2: Collect and treat bacteria.
Transfer bacteria to culture flasks and spin down. 5000 x g, 4C, 10 mins.
Pour out supernatant.
Gently rinse flasks and pellet with small aliquot of TFB1 to remove all traces of media.
Add 100 mL TFB1 per 250 mL of growth culture and resuspend using 10ml serological pipette.
Incubate in wet ice 5 minutes.
Spin down 5000 x g, 4C, 5 mins.
Remove all supernatant.
Add 10 mL TFB2 per 250 mL growth culture and gently resuspend by serological pipette.
Incubate in wet ice 15-60 mins.
Step 3: Dispense and freeze bacteria
Having another person will be helpful at this point
Dispense 100 ul into pre-chilled 1.5 mL microcentrifuge tubes and snap freeze in LN2.
Store at -80C in freezer boxes without dividers.
