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Star Republic: Guide for Biologists

Yeast transformation (II)

1. Grow 10 ml culture in YPD medium for each transformation to a density of 2-5x 107 cells/ml.

2. Spin down cells.

3. Resuspend cells in 20 ml of sterile ddH2O.

4. Spin down cells and resuspend the cells in 100 ml of 100 mM Lithium acetate in 1X TE per 10 ml culture.

5. Pellet cells and resupend the cells in 100 ml of 100 mM Lithium acetate in 1X TE per 10 ml culture.

6. Use 100 ml cells for each transformation.

7. Add transforming DNA (1-5 mg).

8. Add 10 ml of denatured salmon sperm DNA (10 mg/ml).

9. Add 600 ml PEG/LiAc/TE (40% PEG 4000, 0.1 M LiAc, 10 mM Tris-HCl pH7.5, 10 mM EDTA).

10. Vortex.

11. Incubate for 30 minutes at 30 C.

12. Heatshock in 42 C heat block for 15 minutes.

13. Plate directly onto appropriate selection medium or spin down cells, resupend in sterile ddH2O and plate.