Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual

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Bibliographic Information

Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual

David C. Amberg, Daniel Burke, Jeffrey Strathern

Cold Spring Harbor Laboratory Press, c2005

2005 ed.

Available at  / 33 libraries

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Note

Rev. ed. of: Methods in yeast genetics / Dan Burke, Dean Dawson, Tim Stearns, 2000 ed. 2000.

Includes bibliographical references

Description and Table of Contents

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.

Table of 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 b2
  • -Galactosidase in Yeast 9. Plate Assay for Carboxypeptidase a5
  • 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

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