YEAST TWO-HYBRID SCREEN WITH LIBRARY AND BAIT
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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.
