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        Yeast Two-Hybrid Screen with Library and Bait

        互联网

        2200

        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 at30℃

        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℃

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