Manipulation of Plasmids from Yeast Cells

Victoria Lundblad1, Heng Zhou2

1 Baylor College of Medicine, Houston, Texas, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.9
DOI:  10.1002/0471142727.mb1309s39
Online Posting Date:  May, 2001
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Abstract

This unit describes several procedures for manipulating plasmids in yeast cells. The first is a general method to segregate autonomously replicating plasmids from cells: plasmidÔÇÉcontaining yeast cells are grown in nonselective medium, and colonies lacking the plasmid are identified by replica plating. The second, plasmid shuffling, represents a specialized version of plasmid segregation that is useful for analyzing the function of essential genes and for identifying conditional lethal mutations in essential genes. The third approach, plasmid gap repair, is based on the efficient homologous recombination characteristics of yeast cells. Plasmid gap repair can be be used as a method to incorporate mutagenized DNA fragments into a yeast plasmid, rescue genomic mutations onto plasmids, or map alleles of a given gene.

     
 
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Table of Contents

  • Basic Protocol 1: Segregation of Plasmids from Yeast Cells
  • Basic Protocol 2: Plasmid Shuffling
  • Basic Protocol 3: Plasmid Gap Repair for Localized Mutagenesis and Allele Repair
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Segregation of Plasmids from Yeast Cells

  Materials
  • Plasmid‐bearing yeast strain
  • YPD or other nonselective liquid medium and plates (unit 13.1)
  • CM dropout plates (unit 13.1
  • Sterile velvets and replica block (units 13.2 & 1.3)
  • Additional reagents and equipment for growth and manipulation of yeast (unit 13.2)

Basic Protocol 2: Plasmid Shuffling

  Materials
  • YCp vector (unit 13.4) bearing a selectable marker other than URA3
  • Dropout plates +Ura (unit 13.1)
  • Plates with medium selective for YCp vector and containing 5‐FOA (unit 13.1)
  • YPD plates (unit 13.1)
  • YIp5 vector (unit 13.4)
  • Additional reagents and equipment for cloning yeast genes by complementation (unit 13.8), subcloning (unit 3.16), mutagenesis (see Chapter ), lithium acetate transformation (unit 13.7), replica plating (unit 13.2), yeast plasmid DNA preparation (unit 13.11), E. coli transformation (unit 1.8), and gene replacement by transplacement (unit 13.10)

Basic Protocol 3: Plasmid Gap Repair for Localized Mutagenesis and Allele Repair

  Materials
  • YRp or YCp plasmid (unit 13.4) with selectable marker
  • Appropriate restriction enzyme(s) (unit 3.1)
  • Yeast strain with mutation corresponding to selectable marker
  • Plates with medium selective for the plasmid marker
  • Additional reagents and equipment for subcloning (unit 3.16), PCR (unit 15.1), yeast transformation (unit 13.7), growth and manipulation of yeast (unit 13.2), and plasmid segregation (unit 13.9)
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Figures

Videos

Literature Cited

Literature Cited
   Boeke, J.D., Trueheart, J., Natsoulis, G., and Fink, G.R. 1987. 5‐fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164‐175.
   Brenner, C., Bevan, A., and Fuller, R.S. 1994. One‐step site‐directed mutagenesis. Methods Enzymol. 244:163‐165.
   Muhlrad, D., Hunter, R., and Parker, R. 1992. A rapid method for localized mutagenesis of yeast genes. Yeast 8:79‐92.
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