- In the late afternoon, transform 1ul “my” plasmid into 10ul BL21 cells, add 80ul SOC medium, and spread them on a agar plate with Ampicillin, Incubate the plate overnight @ 37°C.
- In the morning, Pick up a colony and plant it in a 5ml LB with Ampicillin, and shake @ 37°C, 250 rpm to the late afternoon.
- Prepare 1L Minimum medium ready (add water and others first, finally add CaCl2, otherwise, precipitation happens!), (just pass through 0.22 um filter, don’t need autoclave) without adding Ampicillin.
- In the late afternoon, spin down the 5ml cells, discard the supernatant, and re-suspend the cells with 50ml Minimum medium with Ampicillin, shake O/N at 37°C, 250 rpm.
- In the morning, transfer the 50 ml culture into 1L minimum medium with Ampicillin (in a 4L big flask), and shake @ 37°C, 250 rpm until O.D. is 0.6 (l = 600nm).
- Add IPTG to 0.5mM, and keep shaking overnight at 18C.
- Spin down the cells at 5000 rpm for 10min, and discard the supernatant, and keep the pellets in -80°C.