标签: Two-Hybrid
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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 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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