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 a30℃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 mliOAc

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

5.Resuspend the pellet in 500μl of 0.1 mliOAc

6.Transformation:

Aliquot 100μl 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:

500μl of 50% PEG 3350

10μl of boiled Herring sperm DNA (place in100℃block for 5 min,then place on ice for 2 min).

72μl of 1 mliOAc

100μl of DNA as noted below

Tube 1: Water only (no DNA)

Tube 2: 1μg of pGAL4 DNA

Tube 3: 40μg of Library DNA

7.Vortex to mix

8.30℃water bath for 30 min,then

42℃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℃

12.quick spin,take off sup

13.Resuspend in the following:

Tube 1: 400μl of water

Tube 2: 400μl of water

Tube 3: 3 ml of water

14.Plate on the following:

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

200μl on a single SMALL Trp/His plate

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

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

400μl on 7 LARGE Leu/Trp/His plates

Invert and Incubate plates for 2-3 days at 30℃