YEAST TWO-HYBRID SCREEN WITH LIBRARY AND BAIT

(The following protocol is for use with the LIBRARY transformation only)

Day 1:

Grow an overnight culture of a single colony of yeast transformed with bait vector in 2.5 ml of SD-Trp medium

Day 2

1. The following morning dilute the overnight culture into 50 ml of YPAD, and grow 4 hours in a 30 C shaker, with vigorous shaking (250-300 rpm)

2. Transfer to a 50 ml Falcon Tube and pellet cells (10 min at 2500K in a clinical centrifuge)

3. Resuspend the pellet in 1 ml of 0.1 M LiOAc

4. Transfer to an Eppendorf tube and spin at top speed for 1 min to pellet cells

5. Resuspend the pellet in 500 ul of 0.1 M LiOAc

6. Transformation:

Aliquot 100 ul of cells into each of 3 eppendorf tubes, quick spin, and take off sup. Then add over the pellet, the following in the following order:

500 ul of 50% PEG 3350

10 ul of boiled Herring sperm DNA (place in 100 C block for 5 min, then place on ice for 2 min).

72 ul of 1 M LiOAc

100 ul of DNA as noted below

Tube 1: Water only (no DNA)

Tube 2: 1 ug of pGAL4 DNA

Tube 3: 40 ug of Library DNA

7. Vortex to mix

8. 30 C water bath for 30 min, then

42 C water bath for 20 min

9. Quick spin to pellet, take off sup.

10. Resuspend pellet in 1 ml of YPAD

11. 30-60 min at 30 C

12. quick spin, take off sup

13. Resuspend in the following:

Tube 1: 400 ul of water

Tube 2: 400 ul of water

Tube 3: 3 ml of water

14. Plate on the following:

Tube 1: 200 ul on a single SMALL Leu/Trp/His plate

200 ul on a single SMALL Trp/His plate

Tube 2: 400 ul on a single LARGE Leu/Trp/His plate

Tube 3: 400 ul on a single LARGE Leu/Trp plate

400 ul on 7 LARGE Leu/Trp/His plates

Invert and Incubate plates for 2-3 days at 30 C.