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Yeast Protocol Handbook

Yeast Protocol Handbook

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Published by Uday Torres Lamus

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Published by: Uday Torres Lamus on Feb 17, 2010
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Yeast Protocols Handbook
PT3024-1 (PR973283)Published July 2009
Yeast Protocols Handbook
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3024-12 Version No. PR973283
I. Introduction 4II. Introduction to Yeast Promoters 5III. Culturing and Handling Yeast 10
IV. Preparation of Yeast Protein Extracts 12
A. General Information 12B. Preparation of Yeast Cultures for Protein Extraction 12C. Preparation of Protein Extracts: Urea/SDS Method 13D. Preparation of Protein Extracts: TCA Method 15E. Troubleshooting 17
V. Yeast Transformation Procedures 18
A. General Information 18B. Reagents and Materials Required 19C. Tips for a Successful Transformation 20D. Integrating Plasmids into the Yeast Genome 20E. Small-scale LiAc Yeast Transformation Procedure 20F. Troubleshooting Yeast Transformation 22 
- and
-Galactosidase Assays 23
A. General Information 23B.
In vivo
Plate Assay Using X-gal in the Medium 26C. Colony-lift Filter Assay 26D. Liquid Culture Assay Using ONPG as Substrate 27E. Liquid Culture Assay Using CPRG as Substrate 28F. Liquid Culture Assay Using a Chemiluminescent Substrate 29G.
-Gal Quantitative Assay 32
VII. Working with Yeast Plasmids 34
A. General Information 34B. Plasmid Isolation From Yeast 34C. Transforming
E. coli 
with Yeast Plasmids 36
VIII. Analysis of Yeast Plasmid Inserts by PCR 39
A. General Information 39B. Tips for Successful PCR of Yeast Plasmid Templates 39
IX. Additional Useful Protocols 42
A. Yeast Colony Hybridization 42B. Generating Yeast Plasmid Segregants 43C. Yeast Mating 44
X. References 46 APPENDICES
A. Glossary of Technical Terms 49B. Yeast Genetic Markers Used in the Matchmaker Systems 51C. Media Recipes 52A. Yeast Media 52B.
E. coli 
Media 55D. Solution Formulations 56E. Plasmid Information 60F. Yeast Host Strain Information 63
Table of Contents
Yeast Protocols Handbook
Protocol No. PT3024-1 www.clontech.com Clontech Laboratories, Inc.Version No. PR973283 3
List of Tables
Table I. Yeast Promoter Constructs Used to Regulate Reporter Gene Expressionin Matchmaker Plasmids and Host Strains 6Table II. Yeast Promoter Constructs in the Matchmaker Cloning Vectors 9Table III. Comparison o
-galactosidase Assays 25Table IV. Selected Yeast Genes and Their Associated Phenotypes 51Table V. Matchmaker Reporter Genes and Their Phenotypes 51Table VI. Matchmaker Two-Hybrid System Cloning Vectors 60Table VII. Matchmaker Two-Hybrid System Reporter and Control Plasmids 61Table VIII. Matchmaker One-Hybrid System Cloning, Reporter & Control Plasmids 62Table IX. Yeast Reporter Strains in the Matchmaker One- and Two-Hybrid Systems 63
List of Figures
Figure 1. Sequence o GAL4 DNA-BD recognition sites in the
 UASs and the UAS
G 17-mer
6Figure 2. Urea/SDS protein extraction method 14Figure 3. TCA protein extraction method 16
Table of Contents
Notice to Purchaser
Clontech products are to be used or research purposes only. They may not be used or any other purpose, including,but not limited to, use in drugs,
in vitro
diagnostic purposes, therapeutics, or in humans. Clontech products may not betranserred to third parties, resold, modifed or resale, or used to manuacture commercial products or to provide a serviceto third parties without written approval o Clontech Laboratories, Inc.Practice o the two-hybrid system is covered by U.S. Patent Nos. 5,283,173, 5,468,614, and 5,667,973 assigned to the ResearchFoundation o the State University o New York. Purchase o any Clontech two-hybrid reagent does not imply or convey alicense to practice the two-hybrid system covered by these patents. Commercial entities purchasing these reagents mustobtain a license rom the Research Foundation o the State University o New York beore using them. Clontech is requiredby its licensing agreement to submit a report o all purchasers o two-hybrid reagents to SUNY Stony Brook. Please contactthe Ofce o Technology Licensing & Industry Relations at SUNY Stony Brook or license inormation (Tel: 631.632.9009;Fax: 631.632.1505).NOTICE TO PURCHASER: LIMITED LICENSEUse o this product is covered by one or more o the ollowing US patents and corresponding patent claims outside theUS: 5,079,352 and 6,127,155. The purchase o this product includes a limited, non-transerable immunity rom suit underthe oregoing patent claims or using only this amount o product or the purchaser’s own internal research. No rightunder any other patent claim (such as method claims in U.S. Patents Nos. 5,210,015, 5,487,972, 5,994,056 and 6,171,785)and no right to perorm commercial services o any kind, including without limitation reporting the results o purchaser’sactivities or a ee or other commercial consideration, is hereby conveyed by the purchase o this product expressly,by implication, or by estoppel. This product is or research use only. Diagnostic uses require a separate license romRoche. Further inormation on purchasing licenses may be obtained by contacting the Director o Licensing, AppliedBiosystems, 850 Lincoln Centre Drive, Foster City, Caliornia 94404, USA.Clontech, the Clontech logo and all other trademarks are the property o Clontech Laboratories, Inc., unless notedotherwise. Clontech is a Takara Bio Company. ©2009 Clontech Laboratories, Inc.
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