Preparation of Yeast DNA

Charles S. Hoffman1

1 Boston College, Chestnut Hill, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.11
DOI:  10.1002/0471142727.mb1311s39
Online Posting Date:  May, 2001
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Abstract

Molecular studies in yeast often require the isolation of both plasmid and chromosomal yeast DNA. Plasmid DNA is used in the transformation of E. coli, whereas chromosomal DNA is used for Southern hybridization analysis, in vitro amplification by the polymerase chain reaction (PCR), or cloning of integrated plasmids. This unit presents two variations of the “smash and grab” protocol that produce suitable DNA for all these applications. These protocols work for both Saccharomyces cerevisiae and Schizosaccharomyces pombe.

     
 
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Table of Contents

  • Section IV: Preparation of Yeast DNA, RNA, and Proteins
  • Basic Protocol 1: Rapid Isolation of Plasmid DNA from Yeast
  • Alternate Protocol 1: Rapid Isolation of Yeast Chromosomal DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Rapid Isolation of Plasmid DNA from Yeast

  Materials
  • YPD or appropriate selective medium (unit 13.1)
  • Yeast colony containing the plasmid of interest
  • recipeBreaking buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (with buffered phenol; unit 2.1)
  • 0.45‐ to 0.52‐mm acid‐washed glass beads (unit 13.12; Thomas Scientific)
  • Competent E. coli cells HB101 or MH1 (unit 1.8)
  • LB plates (unit 1.1) containing appropriate antibiotic (unit 1.4)
  • 13 × 100–mm glass tubes, sterile
  • 30°C incubator with shaker or roller drum
  • Additional reagents and equipment for growth of E. coli on solid medium (unit 1.3) and transformation of E. coli (unit 1.8)

Alternate Protocol 1: Rapid Isolation of Yeast Chromosomal DNA

  • TE buffer ( appendix 22)
  • 1 mg/ml DNase‐free RNase A (unit 5.5)
  • 4 M ammonium acetate solution ( appendix 22)
  • 100% ethanol
  • 18 × 150–mm glass culture tubes or 17 × 100–mm disposable polypropylene tubes, sterile
  • Tabletop centrifuge
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Figures

Videos

Literature Cited

Literature Cited
   Carle, G.F. and Olson, M.V. 1987. Orthogonal‐field‐alternation gel electrophoresis. Methods Enzymol. 155:468‐482.
   Cryer, D.R., Eccleshal, R., and Marmur, J. 1975. Isolation of yeast DNA. Methods Cell Biol. 12:39‐44.
   Davis, R.W., Thomas, M., Cameron, J., St. John, T.P., Scherer, S., and Padgett, R.A. 1980. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 65:404‐411.
   Hall, M.N., Hereford, L., and Herskowitz, I. 1984. Targeting of E.coli β‐galactosidase to the nucleus of yeast. Cell 36:1057‐1065.
   Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557‐580.
   Hoffman, C.S. and Winston, F. 1987. A ten‐minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 27:267‐272.
   Winston, F., Chumley, F., and Fink, G.R. 1983. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 101:211‐228.
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