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(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.