酵母单杂交系统及其实验操作.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 One-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element Protein-DNA interaction The differences between yeast one two hybrid II. Yeast Molecular Techniques Yeast strains Yeast strains C. Mating type compatibilities Y187 (MATα) can mate with AH109, HF7c, CG-1945, Y190, or SFY526 (all MATa). D. Reporter genes Yeast strains E. Leaky HIS3 expression 3-AT : 3-amino-1,2,4-triazole is a competitive inhibitor of the yeast HIS3 protein (His3p).3-AT is used to inhibit low levels of His3p expressed in a leaky manner and thus to suppress background growth on SD medium lacking histidine. Some yeast strains have relatively high basal levels of His3p. Main difference between yeast and E.coli culture To optimize the 3-AT concentration in your selection medium: YEAST MEDIA A. YPD(YPDA) medium 20 g/L Difco peptone 10 g/L Yeast extract 20 g/L Agar (for plates only) (To 0.003% 0.2% adenine hemisulfate Solution) pH to 6.5 To 2% glucosesterile (40% stock solution) YEAST MEDIA B.SD medium(1L) 6.7 g Yeast nitrogen base without amino acids 20 g Agar (for plates only) 850 ml H2O pH to 5.8 100 ml of the appropriate sterile 10X Dropout Solution 3-AT(1 M 3-AT stock solution) To 2% glucosesterile (40% stock solution) C.10X Dropout (DO) Solution Nutrient 10X Concentration L-Adenine hemisulfate salt 200 mg/L L-Arginine HCl 200 mg/L L-Histidine HCl monohydrate 200 mg/L L-Isoleucine 300 mg/L L-Leu