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Pulsed-Field Gel Electrophoresis of Bacterial Chromosomes

  • Julia S. P. Mawer
  • David R. F. Leach
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1054)

Abstract

The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

Key words

Escherichia coli Agarose gel electrophoresis Pulsed-field gel electrophoresis (PFGE) Contour-clamped homogeneous electric field (CHEF) Pulse time Agarose plugs 

Notes

Acknowledgements

We would like to thank Dr. Martin White and Ewa Okely for helpful suggestions to the manuscript.

References

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Julia S. P. Mawer
    • 1
  • David R. F. Leach
    • 1
  1. 1.Institute of Cell BiologyUniversity of EdinburghEdinburghUK

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