Methods in Yeast Genetics and Genomics A Cold Spring Harbor Laboratory Course Manual, 2015 Edition

by Dunham, Maitreya J.; Gartenberg, Marc R.; Brown, Grant W.

• ISBN13: 9781621821342
• ISBN10: 162182134X
• Format: Hardcover
• Copyright: 2015-08-26
• Publisher: Cold Spring Harbor Laboratory Press

Summary

Methods in Yeast Genetics is a course that has been offered annually at Cold Spring Harbor Laboratory for the last 45 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 (2005), revolutionary advances in genomics, proteomics, and imaging technologies have had a significant impact on the field. The 11 experiments included in this manual provide a foundation of methods for any modern-day yeast lab. These methods emphasize combinations of classical and modern genetic approaches, including isolation and characterization of mutants, two-hybrid analysis, tetrad analysis, complementation, and recombination. Also covered are molecular genetic techniques for genome engineering. Additional experiments introduce fundamental techniques in yeast genomics, including both performance and interpretation of Synthetic Genetic Array analysis, multiplexed whole genome and barcode sequencing, and comparative genomic hybridization to DNA arrays. Comparative genomics is introduced using different yeast strains to study natural variation, evolution, and quantitative traits. This manual covers the full repertoire of genetic approaches needed to dissect complex biological problems in the yeast Saccharomyces cerevisiae.

Table of Contents

(preliminary)

Acknowledgements
Preface
A Few Words on Sterile Technique
Yeast Strains
Yeast Plasmids
Yeast Nomenclature
Plates and Media
Yeast Resources
Experiments
Techniques & Protocols

Experiments

1. Transformation of Plasmids & Integrating DNAs
2. Looking at Yeast Cells
3. Manipulating Mating-Type and Epigenetic Transcriptional Silencing
4. Meiosis & Tetrad Dissection
5. Isolation and Characterization of Auxotrophic, Temperature-sensitive, and Osmotic-sensitive Mutants
6. Working with Essential Genes
7. Synthetic Lethal Mutants & Random Sporulation
8. Measuring Mutation Rates and Studying Human Genetic Variation in Yeast
9. Mutation Detection using Comparative Genomic Hybridization
10. Mutation Detection using Whole Genome Sequencing and Linkage
11. Barcode Sequencing and Comparative Functional Genomics

Techniques & Protocols

#1 Genomic Modifications with PCR Products
#2 High Efficiency Yeast Transformation
#3 Using the C6 Cytometer
#4 Modified Hoffman-Winston Genomic DNA Preparation
#5 Indirect Immunofluorescence Microscopy
#6 Yeast Vital Stains
#7 Actin Staining in Fixed Yeast Cells
#8 Preparation of Slides with Agarose Pads for Imaging of Live
Yet Immobile Yeast
#9 Sporulation and Tetrad Dissection
#10 Making a Tetrad Dissection Needle
#11 EMS Mutagenesis
#12 Counting Yeast Cells with a Hemocytometer
#13 Flow Cytometry of Yeast DNA Content Using SYBR Green
#14 Storage and Handling of the Systematic Deletion Collection
#15 Scoring SGA Screens with SGATools
#16 Measuring DNA Concentration with the Qubit Fluorometer
#17 Colony PCR
#18 Training for the Plate Race

Appendices

Appendix A - Media
1. Rich media
2. Synthetic media
3. Sporulation media
4. Indicator media
5. Drug selection media
6. Synthetic Genetic Array (SGA) media
Appendix B - Tetrad Dissection Sheets
Appendix C - 96-well Plate Template
Appendix D - Templates for Making Streaks and Patches