- Lyticase [or Zymolase 100T (ICN, USA)]
- Brij 58
- 50 mM Tris-HCl pH 8.0
- 10% sorbitol
- 100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
- 1 mg/ml leupeptin in water
- 1 mg/ml pepstatin A in methanol
- 100 mM spermidine (pH 7.8)
- 100 mM EDTA
- 1 M MgCl2.
- 4 M KCl
|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).|
Thaw the frozen suspension is thawed rapidly and place it on ice.
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.
|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).|