Introduction of DNA into Yeast Cells

Daniel M. Becker1, Victoria Lundblad2

1 Stanford Law School, Stanford, California, 2 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.7
DOI:  10.1002/0471142727.mb1307s27
Online Posting Date:  May, 2001
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Abstract

The most commonly used yeast transformation protocol is the lithium acetate procedure (described here). It is reasonably fast and provides a transformation efficiency of 105 to 106 transformants/μg. This efficiency rivals that achieved for most, but not all, strains with the more difficult and time‐consuming spheroplast procedure presented here. However, the fastest and easiest of the transformation methods is electroporation, as described in this unit. For a number of strains, electroporation offers the highest transformation efficiency, and may prove especially useful with limiting quantities of transforming DNA. Unlike the lithium acetate procedure, however, electroporation saturates at low DNA levels, restricting its general utility.

     
 
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Table of Contents

  • Basic Protocol 1: Transformation Using Lithium Acetate
  • Alternate Protocol 1: Spheroplast Transformation
  • Alternate Protocol 2: Transfromation by Electroporation
  • Support Protocol 1: Preparation of Single‐Stranded High‐Molecular‐ Weight Carrier DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Transformation Using Lithium Acetate

  Materials
  • YPD medium (unit 13.1)
  • Yeast strain to be transformed
  • YPAD medium: YPD medium supplemented with 30 mg/liter adenine hemisulfate
  • recipeHighest‐quality sterile H 2O
  • 10× TE buffer, pH 7.5 (modify 1× recipe in appendix 22), sterile
  • 10× lithium acetate stock solution: 1 M lithium acetate, pH 7.5 (adjust pH with dilute acetic acid), filter sterilized
  • DNA: high‐molecular‐weight, single‐stranded carrier DNA (see protocol 4support protocol) and transforming DNA
  • 50% (w/v) PEG 4000 or 3350 (do not use PEG 8000), filter sterilized
  • CM dropout plates (unit 13.1) prepared with Difco agar
  • 30°C incubator with shaker
  • Sorvall GSA and SS‐34 rotors (or equivalents)
  • 42°C water bath

Alternate Protocol 1: Spheroplast Transformation

  Materials
  • YPD medium and plates (unit 13.1)
  • Yeast strain to be transformed
  • 1 M sorbitol
  • 2‐mercaptoethanol (2‐ME)
  • Glusulase ( Du Pont NEN)
  • recipeCaCl 2 solution
  • recipeSorbitol/CaCl 2 solution
  • DNA: transforming DNA and 5 mg/ml carrier DNA (sheared calf thymus or salmon sperm DNA; unit 6.3)
  • recipePEG/CaCl 2 solution
  • recipeSelective regeneration agar
  • CM drop‐out plates (unit 13.1)
  • 30°C incubator with rotating platform
  • 55°C water bath
NOTE: Before starting, melt selective regeneration agar (microwave at low setting), aliquot 10‐ml samples into sterile glass test tubes, and place in a 55°C water bath.

Alternate Protocol 2: Transfromation by Electroporation

  Additional Materials
  • 1 M dithiothreitol (DTT; filter sterilize and store at −20°C)
  • 1 M sorbitol
  • recipeSorbitol selection plates
  • Gene Pulser with Pulse Controller ( Bio‐Rad) or Cell‐Porator ( GIBCO/BRL)
  • 0.2‐cm‐gap disposable electroporation cuvettes ( Bio‐Rad) or 0.15‐cm‐gap microelectroporation chambers ( GIBCO/BRL); ice‐cold

Support Protocol 1: Preparation of Single‐Stranded High‐Molecular‐ Weight Carrier DNA

  Materials
  • DNA (type III sodium salt from salmon testes; Sigma #D1626)
  • 1× TE buffer, pH 8.0 ( appendix 22)
  • Buffered phenol (unit 2.1)
  • 1:1 (v/v) phenol/chloroform
  • Chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol, ice‐cold
  • Probe sonicator
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and phenol extraction and ethanol precipitation of DNA (unit 2.1)
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Figures

Videos

Literature Cited

Literature Cited
   Becker, D.M. and Guarente, L. 1991. High‐efficiency transformation of yeast by electroporation. Methods Enzymol. 194:182‐187.
   Beggs, J.D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature (Lond.) 275:104‐109.
   Hinnen, A., Hicks, J.B., and Fink, G.R. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. U.S.A. 75:1929‐1933.
   Ito, H., Fukuda, Y., Murata, K., and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163‐168.
   Lorow‐Murray, D. and Jessee, J. 1991. High‐efficiency transformation of Saccharomyces cerevisiae by electroporation. Focus 13:65‐67.
   Meilhoc, E., Masson, J.‐M., and Teissie, J. 1990. High efficiency transformation of intact yeast cells by electric field pulses. Bio/Technology 8:223‐227.
   Orr‐Weaver, T.L., Szostak, J.W., and Rothstein, R.J. 1983. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 101:228‐244.
   Schiestl, R.H. and Gietz, R.D. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16:339‐346.
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