酵母遗传实验方法 冷泉港实验课手册 英文版PDF电子书下载

Experiments 1

Ⅰ.Looking at Yeast Cells 1

Ⅱ.Isolation and Characterization of Auxotrophic,Temperaturesensitive,and UV-sensitive Mutants 7

Ⅲ.Meiotic Mapping 17

Ⅳ.Mitotic Recombination and Random Spore Analysis 29

Ⅴ.Transformation of Yeast 39

Ⅵ.Synthetic Lethal Mutants 49

Ⅶ.Gene Replacement 55

Ⅷ.Isolation of ras2 Suppressors 73

Ⅸ.Manipulating Cell Types 79

Ⅹ.Isolating Mutants by Insertional Mutagenesis 89

Ⅺ.Two-hybrid Protein Interaction Method 97

Techniques and Protocols 103

1.High-efficiency Transformation of Yeast 103

2.“Lazy Bones”Plasmid Transformation of Yeast Colonies 107

3.Yeast DNA Isolations 109

A.Yeast DNA Miniprep(40 ml) 109

B.Yeast DNA Miniprep(5 ml) 110

C.A Ten-minute DNA Preparation from Yeast 111

D.Yeast Genomic DNA:Glass Bead Preparation 113

4.Yeast Protein Extracts 115

5.Yeast RNA Isolation 117

6.Hydroxylamine Mutagenesis of Plasmid DNA 121

7.Assay of β-Galactosidase in Yeast 123

8.Plate Assay for Carboxypeptidase Y 127

9.Random Spore Analysis 129

10.Yeast Vital Stains 131

11.Yeast Immunofluorescence 133

12.Actin Staining in Fixed Cells 137

13.PCR Protocol for PCR-mediated Gene Disruption 139

14.Yeast Colony PCR Protocol 141

15.Measuring Yeast Cell Density by Spectrophotometry 143

16.Cell Synchrony 145

17.Chromatin Immunoprecipitation 149

18.Flow Cytometry of Yeast DNA 155

19.Logarithmic Growth 157

20.EMS Mutagenesis 159

21.Tetrad Dissection 161

22.Making a Tetrad Dissection Needle 165

23.Picking Zygotes 167

24.Determining Plating Efficiency 169

Appendices 171

A.Media 171

B.Stock Preservation 183

C.Yeast Genetic and Physical Maps 185

D.Templates for Making Streak Plates 193

E.Electrophoretic Karyotypes of Strains for Southern Blot Mapping 195

F.Strains 197

G.Counting Yeast Cells with a Standard Hemocytometer Chamber 199

H.Tetrad Scoring Sheet 201

I.Trademarks 203

J.Suppliers 205