|1. ||Resuspend the cells in chilled lysis buffer in a ratio of 1 g cell wet weight to 1 ml lysis buffer. |
- Add the PMSF (10 µl PMSF (100 mM) per ml of celsuspension) at this point.
|2. ||Add lysosyme to a final concentration of 300 µg/ml and incubate the cell suspension at 4°C for 4 h. |
|3. ||Add 5 µl MgCl2 (1 M) and 1 µl DNase solution (1 mg/ml) per ml of cell suspension and incubate the solution at 4°C for 30 min. |
|4. ||Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman). |