Direct PCR of Intact Bacteria (Colony PCR)

Michael E. Woodman1

1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3D
DOI:  10.1002/9780471729259.mca03ds9
Online Posting Date:  May, 2008
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Abstract

This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. Curr. Protoc. Microbiol. 9:A.3D.1-A.3D.6. © 2008 by John Wiley & Sons, Inc.

Keywords: colony PCR; bacteria; screen

     
 
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Table of Contents

  • Basic Protocol: PCR Amplification of DNA from a Bacterial Colony (Colony PCR)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: PCR Amplification of DNA from a Bacterial Colony (Colony PCR)

 Materials
  • 10× PCR buffer: supplied with DNA polymerase or see appendix 2A
  • 2.5 mM (each) dNTP mix: supplied with DNA polymerase or see appendix 2A
  • 30 µM oligonucleotide primer 1 stock (see Critical Parameters)
  • 30 µM oligonucleotide primer 2 stock (see Critical Parameters)
  • 5 U/µl Taq DNA polymerase, heat-stable (e.g., Takara Taq, Fisher)
  • Distilled H2O, sterile
  • MgCl2 (if required)
  • Gel electrophoresis loading buffer (see recipe)
  • 200-µl thin-walled PCR tubes (or other appropriate for thermal cycler)
  • Toothpicks, sterile
  • Thermal cycler
  • Additional reagents and equipment for performing agarose (Voytas, 2000) or polyacrylamide (Chory and Pollard, 1999) gel electrophoresis
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Figures

  •  FigureFigure A.3D.1 Representative results of colony PCR. Lane 1: 1-kb ladder (New England BioLabs); Lane 2: ~1.3-kb insert; Lane 3: ~400-bp insert; Lane 4: No insert. The faint bands at the bottom of the gel are unused primers from the PCR reaction.
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  •  FigureFigure A.3D.2 Diagram of oligonucleotide primer locations for colony PCR. Primers 1 + 4 determine if a DNA insert size is correct. Primers 1 + 3 or primers 2 + 4 can be used to determine if there is a DNA insertion as well as its orientation. For additional confirmation of the orientation of the insert, PCR reactions can be performed with primers 1 + 2 or primers 3 + 4 as well (see Anticipated Results).
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Literature Cited

Literature Cited
    Chory, J. and Pollard, J.D. Jr. 1999. Separation of small DNA fragments by conventional gel electrophoresis. Curr. Protoc. Mol. Biol. 47: 2.7.1-2.7.8.
    Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56: 15.1.1-15.1.14.
    Riley, S.P., Woodman, M.E., and Stevenson, B. 2008. "Culture of Escherichia coli and related bacteria". In Current Protocols Essential Laboratory Techniques (S.R. Gallagher and E.A. Wiley, eds.) pp. 4.2.1-4.2.25. John Wiley & Sons, Hoboken, N.J.
    Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51: 2.5A.1-2.5A.9.
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