© 2005 • 230 pp., illus., index
Paperback • $96.00 • ISBN  978-087969728-0

“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.

  “This update of the 2000 edition is the latest version of a book that remains the best single source of basic genetics techniques for Saccharomyces cerevisiae.... Based on the long running course at Cold Spring Harbor, it contains a mixture of experiments, techniques, protocols and basic data. The experiments include very clear explanations of the logic behind the approaches. New techniques like TAP tagging are included and many familiar experiments have been revised to take into account, for example, cytological advances with better fluorophores and GFP–fusions, and the ready availability of systematic deletion strains. It is an essential purchase for all laboratories.”
      —Microbiology Today

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 β-Galactosidase in Yeast

9. Plate Assay for Carboxypeptidase Υ

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