Continue Expression of MBP-intein.
- After overnight growth, spin cells at 4500rpm at 4°C for 15 minutes.
- Resuspend each of the pellets in 1mL of LB broth with glucose(taken from 1L sterile LB with glucose 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 with glucose.
- 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 2.5hours.
- 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).
- Freeze cells with liquid nitrogen.
- Store at -80°C.
- The OD600 was 1.3 when the cells were induced with IPTG.