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.