Methods in Yeast Genetics A Laboratory Course Manual

About this book

Yeast is an ideal eukaryotic microorganism for biochemical and genetic studies, particularly since its complete genome sequence became available. The Cold Spring Harbor Laboratory Yeast Genetics Course has kept pace with the field over the last 30 years, combining essential standard methods with the latest technologies, and its course manual has followed suit. In the new 2000 edition, most of the experiments have been revised and several new experiments and techniques have been added, including the 2-hybrid system, PCR-based methods, and isolation of chromatin. This manual remains a primary source for newcomers to the field as well as the established investigator, providing complete, detailed protocols and techniques of proven robustness.

Contents

Experiments: looking at yeast cells; isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants; meiotic mapping; mitotic recombination and random spore analysis; transformation of yeast; synthetic lethal mutants; gene replacement; isolation of ras2 suppressors; manipulating cell types; isolating mutants by insertional mutagenesis; two-hybrid protein interaction method. Techniques and protocols: high-efficiency transformation of yeast; "Lazy Bones" plasmid transformation of yeast colonies; yeast DNA isolations; yeast DNA miniprep (40 ml); yeast DNA miniprep (5 ml); a ten-minute DNA preparation from yeast; yeast genomic DNA - glass bead preparation; yeast protein extracts; yeast RNA isolation; hydroxylamine mutagenesis of plasmid DNA; assay of beta-galactosidase in yeast; plate assay for carboxypeptidase Y; random spore analysis; yeast vital stains; yeast immunofluorescence; actin staining in fixed cells; PCR protocol for PCR-mediated gene disruption; yeast colony PCR protocol; measuring yeast cell density by spectrophotometry; cell synchrony; chromatin immunoprecipitation; flow cytometry of yeast DNA; logarithmic growth; EMS mutagenesis; tetrad dissection; making a tetrad dissection needle; picking zygotes; determining plating efficiency. Appendices: media; stock preservation; yeast genetic and physical maps; templates for making streak plates; electrophoretic karyotypes of strains for southern blot mapping; strains; counting yeast cells with a standard hemocytometer chamber; tetrad scoring sheet; trademarks; suppliers.

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