Direct PCR of Intact Bacteria (Colony PCR)

Michael E. Woodman1, Christina R. Savage2, William K. Arnold2, Brian Stevenson2

1 Present address: Michael E. Woodman, Eli Lilly and Company, Indianapolis, Indiana, 2 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/cpmc.14
Online Posting Date:  August, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by John Wiley & Sons, Inc.

Keywords: colony PCR; bacteria; screen

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: PCR Amplification of DNA from a Bacterial Colony (Colony PCR)
  • Alternate Protocol 1: Colony PCR: Enhancement of Efficiency
  • REAGENTS AND SOLUTIONS
  • COMMENTARY
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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 Ex Taq DNA polymerase, Takara or Platinum Taq DNA polymerase, Thermo Fisher Scientific)
  • Distilled H 2O, sterile
  • MgCl 2 (if required)
  • Gel electrophoresis loading buffer (see recipe)
  • 200‐μl thin‐walled PCR tubes (or other appropriate tubes for thermal cycler)
  • Toothpicks, sterile
  • Thermal cycler
  • Additional reagents and equipment for performing agarose gel electrophoresis
  • (Voytas, ) or polyacrylamide gel electrophoresis (Chory and Pollard, )

Alternate Protocol 1: Colony PCR: Enhancement of Efficiency

  Additional Materials (also see protocol 1Basic Protocol)
  •   Distilled water
  • 500‐μl microcentrifuge tubes
  • Water bath (boiling) or 100°C heat block
  • Microcentrifuge
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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. doi: 10.1002/0471142727.mb0207s47.
  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. doi: 10.1002/0471142727.mb1501s56.
  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. doi: 10.1002/0471142727.mb0205as51.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library