Methods in Yeast Genetics. 2005 Edition

Description

“Methods in Yeast Genetics” is a course that has been offered annually at Cold Spring Harbor Laboratory for the last thirty years. This is an updated edition of the course manual, which provides a set of teaching experiments, along with protocols and recipes for the standard techniques and reagents used in the study of yeast biology. Since the last edition of the manual was published (2000), revolutionary advances in genomics and proteomics technologies have had a significant impact on the field. This updated edition reflects these advances, and also includes new techniques involving vital staining, visualization of Green Fluorescent Protein, new drug resistance markers, high-copy suppression, Tandem Affinity Protein tag protein purification, gene disruption by double-fusion polymerase chain reaction, and many other recent developments.

Contents

EXPERIMENTS

• I. 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 ras2 Suppressors
• 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 PROTOCOLS

• 1. 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 ß-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 DiOC6 or 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. coli
• 27. Preparing and Transforming Competent E. coli
• 28. Storing and Handling of the Systematic Deletion Collection
• APPENDICES