EMS and UV Mutagenesis in Yeast

Fred Winston1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.3B
DOI:  10.1002/0471142727.mb1303bs82
Online Posting Date:  April, 2008
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Abstract

Many fundamental biological processes have been elucidated by the isolation and analysis of mutants that are defective in such processes. Therefore, the methods to generate mutants are of great importance in model organisms. This unit describes two protocols for mutagenesis of yeast—using ethyl methanesulfate (EMS) and ultraviolet (UV) light. Each of these methods has been used successfully for many years. Curr. Protoc. Mol. Biol. 82:13.3B.1‐13.3B.5. © 2008 by John Wiley & Sons, Inc.

Keywords: yeast; mutagenesis; EMS; UV

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Mutagenesis Using Ethyl Methanesulfonate (EMS)
  • Alternate Protocol 1: Mutagenesis Using UV Irradiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Mutagenesis Using Ethyl Methanesulfonate (EMS)

  Materials
  • Desired yeast strain
  • YPD medium and plates (unit 13.1)
  • Sterile water
  • 0.1 M sodium phosphate buffer, pH 7.0 (see recipe)
  • Ethyl methanesulfonate (EMS; Kodak)
  • 5% (w/v) sodium thiosulfate (Sigma), autoclaved for sterility
  • 13 × 100–mm culture tube
  • Vortex
  • 30°C incubator with rotating platform
  • Canavanine plates (unit 13.1)
  • Additional reagents and equipment for growing cells, determining cell density, and replica plating (unit 13.2) and for storage of strains (unit 13.1)
CAUTION: EMS is a dangerous mutagen. All solutions, plasticware, and glassware that come into contact with EMS should be rinsed with 5% sodium thiosulfate to inactivate the EMS.

Alternate Protocol 1: Mutagenesis Using UV Irradiation

  • UV germicidal light bulb (Sylvania G15T8; 254 nm wavelength) or Stratagene UV Crosslinker
  • UV dosimeter (optional)
  • 23°C incubator
CAUTION: Wear safety glasses to protect eyes from UV light.
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Figures

Videos

Literature Cited

Literature Cited
   Botstein, D. and Jones, E.W. 1969. Nonrandom mutagenesis of the Escherichia coli genome by nitrosoguanidine. J. Bacteriol. 98: 847‐848.
   Coulondre, C. and Miller, J.H. 1977. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J. Mol. Biol. 117: 577‐606.
   Muster‐Nassal, C. and Kolodner, R. 1986. Mismatch correction catalyzed by cell‐free extracts of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 83: 7618‐7622.
   Prakash, L. and Sherman, F. 1973. Mutagenic specificity: Reversion of iso‐1‐cytochrome c mutants of yeast. J. Mol. Biol. 79: 65‐82.
   Rose, M.D., Winston, F., and Hieter, P. 1990. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
   Siede, W. and Eckardt‐Schupp, F. 1986. A mismatch repair‐based model can explain some features of UV mutagenesis in yeast. Mutagenesis 1: 471‐474.
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