Continue expression of of BL21(DE3) E.coli with mutated GFP plasmid (D1C).
- After overnight growth, spin cells at 4500rpm at 4°C for 15 minutes.
- Resuspend each of the pellets in 1mL of LB broth (taken from 1L sterile LB previously made in 2800mL flasks).
- Add each of the resuspended pellets with broth to one of the four 2800mL flasks previously made with 1L of sterile LB.
- Add 1mL 100mg/mL ampicillin to each flask.
- 100mg/mL ampicillin prepared fresh today with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
- Incubate these four flasks at 37°C at 165rpm until 0.6 OD600 is reached.
- Add 1mL 0.1M IPTG to each flask and and continue growth for 2.5 hours.
- Spin cells at 4500rpm at 4°C for 15 minutes.
- Resuspend cells in column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA).
- Store at -20°C.