Spheroplast Isolation

1. Spin down cells.

2. Resuspens cells in STE solution (0.5 M sucrose, 10 mM Tris–HCl, and 10 mM EDTA, pH 8.0).

4. Incubation with rotating mixing at 25°C for 30 min.

5. Spin down cells 12,000×g for 1 min.

6. Wash with solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8). Spin down

7. Resuspend the pellet in solution A containing 1 mg/ml of hen egg lysozyme.

8. Incubate at 25 °C for 15 ~30 min.

9. Spin 12,000×g for 5 min.

10 Resuspend the spheroplasts in TE buffer