- Inoculate 25 ml of sterile culture medium containing both 25 μg/ml chloramphenicol and 50 μg/ml kanamycin in a 125 ml Erlenmeyer flask.
- After growing the cultures overnight, inoculate a 2 L Erlenmeyer flask containing 400 ml of sterile media with antibiotics with 20 ml of the overnight culture. Inoculate a 250 ml Erlenmeyer flask containing 50 ml of sterile media and antibiotics with 2.5 ml of the overnight culture. This is your negative control.
- Grow cultures at 37°C with vigorous shaking.
- Measure and record the OD600 of the 2L cultures every 15-20 mins.
- Once the OD600 is 0.6, induce the culture in the 2 L flask by adding IPTG to a nal concentration of 0.05 mM. Do not induce the negative control!
- Return the 2 L flasks to the shaking incubator.
- Measure and record the OD600 of both the induced culture and the negative control every hour.
- After 4-5 hours, transfer a 1 ml sample from each Erlenmeyer flask to a labeled 1.5 ml microcentrifuge tube.
- Pellet the cells by centrifugation at 4000 g for 20 mins.
- Discard the supernatant into a waste container and store the cells overnight at 20°C.
- Allow the rest of the flasks to grow overnight.
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