Expressing the Double Mutant
- Centrifuge the cultures grown overnight at 4500rpm at 4°C for 15 minutes.
- Resuspend each of the pellets in 1mL of LB broth.
- 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.
- Add 1mL of 40mg/ml hemin in dimethylformamide to each of the flasks.
- Incubate these flasks at 37°C at 135rpm for about three hours.
- Add 1mL of 0.1M IPTG to each flask. Continue to incubate at 37°C at 135rpm for about five more hours.
- Centrifuge these cells at 4500rpm at 4°C for 15 minutes.
- Resuspend the pellets in 50mM Tris 50mM NaCl, pH ~9.
- Place resuspended cells in -80°C freezer for long-term storage.