|1. ||Resuspend the cells in chilled lysis buffer. Normally ratios of cell wet weight to buffer volume of 1:1 to 1:4 are used. |
|2. ||Cool the cell suspension on ice for 10 min. |
- Add 10 µl PMSF (100 mM) per ml of celsuspension after this step.
|3. ||Sonicate the cell suspension with 10 short burst of 10 sec followed by intervals of 30 sec for cooling. |
- Keep the suspension at all times on ice.
- Avoid foaming.
- Dont go away while the sonicator is in operation. It is possible that the beaker breaks or turns in the melting ice.
|4. ||Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman). |