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Table I. Yeast Promoter Constructs Used to Regulate Reporter Gene Expression in Matchmaker Plasmids and Host Strains Yeast Promoter Constructs in the Matchmaker Cloning Vectors Comparison of β-galactosidase Assays Selected Yeast Genes and Their Associated Phenotypes Matchmaker Reporter Genes and Their Phenotypes Matchmaker Two-Hybrid System Cloning Vectors Matchmaker Two-Hybrid System Reporter and Control Plasmids Yeast Reporter Strains in the Matchmaker One- and Two-Hybrid Systems
6 9 25 52 52 61 62 63 64
Table II. Table III. Table IV. Table V. Table VI. Table VII. Table IX.
Table VIII. Matchmaker One-Hybrid System Cloning, Reporter & Control Plasmids
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Protocol No. PT3024-1 Version No. PR742227
Yeast Protocols Handbook
Table of Contents continued List of Tables
6 14 16
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VIII. Analysis of Yeast Plasmid Inserts by PCR A. General Information B. Tips for Successful PCR of Yeast Plasmid Templates IX. Additional Useful Protocols A. Yeast Colony Hybridization B. Generating Yeast Plasmid Segregants C. Yeast Mating
Yeast Protocols Handbook
FOR RESEARCH USE ONLY
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PT3024-1 (PR742227) Published 11 February 2008
Yeast Protocols Handbook
List of Figures
Figure 1. Figure 2. Figure 3.
Sequence of GAL4 DNA-BD recognition sites in the GAL1, GAL2, MEL1 UASs and the UASG 17-merHale Waihona Puke Baidu Urea/SDS protein extraction method TCA protein extraction method
Table of Contents
I. Introduction II. Introduction to Yeast Promoters III. Culturing and Handling Yeast IV. V. VI. VII. Preparation of Yeast Protein Extracts A. General Information B. Preparation of Yeast Cultures for Protein Extraction C. Preparation of Protein Extracts: Urea/SDS Method D. Preparation of Protein Extracts: TCA Method E. Troubleshooting Yeast Transformation Procedures A. General Information B. Reagents and Materials Required C. Tips for a Successful Transformation D. Integrating Plasmids into the Yeast Genome E. Small-scale LiAc Yeast Transformation Procedure F . Troubleshooting Yeast Transformation α- and β-Galactosidase Assays A. General Information B. In vivo Plate Assay Using X-gal in the Medium C. Colony-lift Filter Assay D. Liquid Culture Assay Using ONPG as Substrate E. Liquid Culture Assay Using CPRG as Substrate F . Liquid Culture Assay Using a Chemiluminescent Substrate G. α-Gal Quantitative Assay Working with Yeast Plasmids A. General Information B. Plasmid Isolation From Yeast C. Transforming E. coli with Yeast Plasmids 4 5 10 12 12 12 13 15 17 18 18 19 20 20 20 22 23 23 25 25 26 27 28 32 34 34 34 36 39 39 39 42 42 43 44 46 49 50 52 53 53 56 57 61 64
X. References XI. Matchmaker and Related Products PPENDICES A A. Glossary of Technical Terms B. Yeast Genetic Markers Used in the Matchmaker Systems C. Media Recipes A. Yeast Media B. E. coli Media D. Solution Formulations E. Plasmid Information F . Yeast Host Strain Information