2. Wash spheroplasts with 1 M sorbitol, 10 mM Tris-HCl pH 7.5, 10 mM CaCl₂.

3. Resuspend spheroplasts in 1 M sorbitol, 10 mM Tris-HCl pH 7.5, 10 mM CaCl₂ at the concentration of 2-5x 10⁸ spheroplasts/ml.

At this stage, spheroplasts can be aliquotted and stored at -70 °C.

4. Use 100 ml spheroplasts per transformation.

5. Add 1-10 mg DNA, incubate at room temperature for 15 minutes.

6. Add 1 ml 10 mM Tris-HCl pH 7.5, 10 mM CaCl₂, 20% PEG 8000.

7. Invert gently to mix. Incubate 10 minutes at room temperature.

8. Centrifuge at 500 g for 5 minutes.

9. Carefully remove supernatant.

10. Resuspend spheroplasts in 0.2-0.5 ml of 1 M sorbitol, 6.5 mM CaCl₂, 0.25% yeast extract, 0.5% Bacto-peptone.

11. Incubate at 30 °C for 30-60 minutes without shaking.

12. Plate 0.2 ml on sorbitol plates (e.g., plates containing minimum medium plus 1 M sorbitol).

13. Transformants appear in 2-5 days at 30 °C.