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 -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