1. Inoculate 1 ml of YPDA or SD with several 2–3 mm colonies.
2. Vortex vigorously to disperse any clumps.
3. Transfer cells to a flask containing 50 ml of YPDA or SD:
4. Incubate at 30oC for 16–18 hr with shaking (250 rpm) to stationary phase (OD600>1.5).
5. Transfer overnight culture (enough to produce an OD600 = 0.2–0.3) into 300 ml of YPDA
6. Incubate at 30oC for 3 hr with shaking (230–270 rpm).
7. Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature.
8. Discard the supernatant and resuspend cell pellets by vortexing in 25–50 ml of sterile TE or H2O
9. Pool cells centrifuge at 1,000 x g for 5 min at room temperature.
10. Decant the supernatant.
11. Resuspend the cell pellet in 1.5 ml of freshly prepared, sterile 1X TE/LiAc:
12. Prepare 10 ml PEG/LiAc solution.
13. In the indicated tube, mix the followinga:
• DNA-BD/bait 0.1 µg
• AD/library 0.1 µg
• Herring testes carrier DNA 0.1 mg (10µl)
14. Add 0.1 ml of yeast competent cells and mix well by vortexing.
15. Add 0.6 ml of sterile PEG/LiAc solution and vortex at high speed to mix.
16. Incubate at 30oC for 30 min with shaking (200 rpm).
17. Add 70 µl of DMSO. Mix well by gentle inversion or swirling. Do not vortex.
18. Heat shock for 15 min in a 42oC water bath.
19. Chill cells on ice for 1–2 min.
20. Centrifuge cells for 5 sec at room temperature at 14 K rpm
21. Remove the supernatant.
22. Resuspend cells in 0.5 ml of 1X TEb:
23. Proceed to plating (on either SD –Trp/–Leu) or SD –Trp/–Leu/–His/–Ade with or without X-á-gal as required.