Large scale yeast transformation/2H

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COHscientist
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Large scale yeast transformation/2H

essentially as described in Clontech 2H manual, for Gal4 based system with His/Leu/Trp selection

1. 4 x 10 ml YPD, each inoculated with single colony of HF7c from a fresh dish. 16h, 30C, shaking.

2. After 16h - transfer to 2 x 200 ml YPD. 30C, shaking, 4h 30 min. Check OD600 - should be around 0.9 - 1.0

3. Pour in 8 x 50 ml Falcons. Centrifuge RT, 5 min 1000 rpm.

4. Wash with dH2O, ~30 ml/Falcon. Pellet as aboove.

5. Resuspend yeast in 8 ml 1 x TE/LiAc.

6. Mix 500 mkg bait plasmid, 250 mkg library plasmid and 50 mg denatured salmon sperm DNA.
Make PEG/LiAc solution - 60 ml (48 ml PEG + 6 ml 10xTE + 6 ml 10xLiAc).

7. Add the DNA mixture to the yeast, then add PEG/LiAc. 30 min shaker, 30C.

8. Add 7 ml DMSO. Heat shock 15 min 42C.

9. Chill 10 min. Pellet yeast 1000 rpm, 5 min; remove supernatant. If doing a stringent screen, go directly to step 11. If nonstringent screen or toxic bait - do step 10, which increases ~20-30 x number of colonies.

10. Resuspend in 20 ml YPD. Shake at 30C, 1 hour.

11. Pellet yeast and resuspend in 5 ml SD.

5 mkl + 95 mkl H2O - plate on SD+His-Leu-Trp (control for cotransformation efficiency)
5 mkl + 95 mkl H2O - plate on SD+His+Trp-Leu (control for library plasmid transformation)

All the rest - plate on ~ 15-20 big dishes, SD-His-Leu-Trp.