Methods in Yeast Genetics 2005 A Cold Spring Harbor Laboratory Course Manual

EXPERIMENTSI.Looking at Yeast Cells II.Isolation and Characterization of Auxotrophic, Temperature-sensitive, and Osmotic-sensitive Mutants III.Meiotic Mapping IV.Mitotic Recombination and Random Spore Analysis V.Transformation of Yeast VI.Synthetic Lethal Mutants VII.Gene Replacement VIII.Isolation of ras2Suppressors IX.Manipulating Cell Types X.Isolating Mutants by Insertional Mutagenesis XI.Isolation of Separation of Function Mutants by Two-hybrid Differential Interaction Screening TECHNIQUES AND PROTOCOLS1.High-efficiency Transformation of Yeast 2.“Quick and Dirty” Plasmid Transformation of Yeast Colonies 3.Yeast DNA Isolations A.Yeast DNA Miniprep (40 ml) B.Yeast DNA Miniprep (5 ml) C.A Ten-minute DNA Preparation from Yeast D.Yeast Genomic DNA: Glass-bead Preparation 4.Yeast Protein Extracts 5.TAP Purification 6.Yeast RNA Isolations 7.Hydroxylamine Mutagenesis of Plasmid DNA 8.Assay of b-Galactosidase in Yeast 9.Plate Assay for Carboxypeptidase ? 10.Random Spore Analysis 11.Yeast Vital Stains A.DAPI Stain of Nuclear and Mitochondrial DNA B.Visualizing Mitochondria with DiOC6or DilC5(3) C.Visualizing Vacuoles and Endocytic Compartments with FM4-64 D.Calcofluor Staining of Chitin and Bud Scars 12.Yeast Immunofluorescence 13.Actin Staining in Fixed Cells 14.PCR-mediated Gene Disruption A.One-step PCR Gene Disruption B.Gene Disruption by Double-fusion PCR 15.Yeast Colony PCR 16.Measuring Yeast Cell Density by Spectrophotometry 17.Cell Synchrony 18.Chromatin Immunoprecipitation 19.Flow Cytometry of Yeast DNA 20.Logarithmic Growth 21.EMS Mutagenesis 22.Tetrad Dissection 23.Making a Tetrad Dissection Needle 24.Picking Zygotes 25.Determining Plating Efficiency 26.DNA Miniprep from E. coli27.Preparing and Transforming Competent E. coli28.Storing and Handling of the Systematic Deletion Collection APPENDICESAmberg, David C. is the author of 'Methods in Yeast Genetics 2005 A Cold Spring Harbor Laboratory Course Manual' with ISBN 9780879697280 and ISBN 0879697288.