酵母单杂交系统和其实验操作.pptVIP

Yeast One Hybrid System;I. Introduction II. Yeast Molecular Techniques III. Constructing a Reporter Vector for One-Hybrid Analysis IV. Generating a cDNA Library V. Constructing Screening a One-Hybrid Library VI. Analyzing Positive Interactions ;I. Introduction ; ;The differences between yeast one two hybrid;授丁射镰巍毖旭湛耐突五团叠郡删籍殴褐谎宗玩济危扎不币妥拂议肄记盟酵母单杂交系统和其实验操作酵母单杂交系统和其实验操作;II. Yeast Molecular Techniques ;Yeast strains;Yeast strain：Y187（MATα） Can’t synthesis His、 Leu、Trp by itself。;Yeast strains;Yeast strains;Main difference between yeast and E.coli culture;To optimize the 3-AT concentration in your selection medium:;YEAST MEDIA;YEAST MEDIA B.SD medium(1L);C.10X Dropout (DO) Solution;Supplied 10 g –Leu DO Supplement(to measure the transformation efficiency of the library plasmid ) 10 g –Trp DO Supplement( to measure the transformation efficiency of the reporter plasmid ) 10 g –Leu/–Trp DO Supplement(to measure the number of clones screened ) 10 g –His/–Leu/–Trp DO Supplement(select for one-hybrid interactions ） Unsupplied –His/–Trp DO Supplement（to measure a pHIS2 reporter plasmid for background growth） ;Preparation of Competent Yeast Cells—LiAc Method;Small-scale LiAc Yeast Transformation Procedure;Notes: For simultaneous cotransformation , use 0.1 mg of each plasmid , in addition to the 0.1 mg of carrier DNA. carrier DNA. V1/5 Glass Air 30℃;Troubleshooting of Yeast Transformation;Troubleshooting of Yeast Transformation; III.Constructing a Reporter Vector for One-Hybrid Analysis ;技 术 路 线;A. Background Identified a true or putative target element A construct composed of one or more tandem copies of your target Constructs should be tested for background (leaky) HIS3 expression before you start a one-hybrid analysis ;B. Synthesize Your Target Element The reporter should contain at least three tandem copies of the DNA target The most convenient and reliable method for generating them to be oligonucleotide synthesis ;;Background HIS3 Expression of ICEr2;IV. Generating a