Day 1: Inoculate overnight culture
Autoclave 2 x 200 ml LB medium.
In the evening, inoculate the medium (containing the appropriate antibiotic) with either a bacterial colony or a chunk of glycerol stock. Grow overnight at 37°C with shaking at 220 rpm.
Day 2: Grow SeMet protein
- Inoculate 10 ml of overnight culture into a fresh medium (200 ml) containing the appropriate antibiotic (e.g. ampicillin at 100 ug/ml final concentration). Grow at 37°C, 220 rpm until the OD600 reaches 0.3.
- While the bacterial cells are growing, prepare the SelenoMet Medium Base (cf. Fig. 1)
- Spin down in table-top centrifuge (e.g. Allegra X-15R (Beckman Coulter)) in 4 x 50 ml falcon tubes.
- Resuspend with 20 ml of ready-to-use SelenoMet Medium Base taken from 1 litre prepared above
- Grow at 37°C, 220 rpm until OD600 = 0.6.
- Induce with IPTG (1 mM final concentration) and transfer to 18°C.
Day 3: Purification of the protein
Refer to protocol for purification of native protein.