|1. ||Resuspend the cells in an equal amount of chilled working buffer. Adjust the pH of the suspension to 8.2 with solid Tris base. |
|2. ||Freeze the suspension rapidly in liquid nitrogen in an ultracentrifuge tube. (Freezing and thawing is essential in this procedure). |
|3. ||Thaw the frozen suspension is thawed rapidly and place it on ice. |
- Add 10 µl PMSF (100 mM) per ml of celsuspension at this point.
|4. ||Add the following solutions to the cell suspension: 4 M KCl to a concentration of 1 M, 100 mM spermidine to 5 mM, and lyticase to 2000 U/ml. |
- Instead of lyticase, Zymolase 100T could be used at a final concentration of 0.5 mg/ml.
|5. ||Mix gently and incubate the suspension for 30 min on ice, with occasional swirling. Usually over 90% of the cells are converted to spheroplasts. |
|6. ||Add different protease inhibitors: 2 µl/ml EDTA (0.5 M), 1 µl/ml PMSF (100mM), 1 µl/ml leupeptin (1 mg/ml), and 1µl/ml pepstatin A (1 mg/ml) to the suspension. |
|7. ||Add Brij 58 to a concentration of 0.1% and heat shock the spheroplasts for 3 min at 37°C. |
|8. ||Remove cell debris by centrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman). |