Purification of Bacteriophage Clones

Thomas Quertermous1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.5
DOI:  10.1002/0471142727.mb0605s13
Online Posting Date:  May, 2001
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Abstract

Careful purification of the clone of interest away from contaminating phage is required before growth and characterization of the clone can proceed. It is common for a “purified” clone to be contaminated by a second phage, leading to confusing results and wasted time. Several rounds of purification should be performed even if the phage appears pure as early as the secondary screening stage. In this unit, phage plates are correctly oriented to the autoradiograph film, and a region that should contain the clone of interest is sampled by toothpicking each phage plaque onto secondary plates containing a lawn of host cells. Alternatively, a plug of agarose can be taken from the primary plate, placed in suspension medium, and this solution used to plate a small secondary library. Plaques on the secondary plates are transferred to nitrocellulose filters, hybridized to a 32P‐labeled probe, and an isolated positive plaque is picked, diluted in suspension medium, and regrown. This process is repeated until the desired plaque is purified.

     
 
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Table of Contents

  • Section III: Purification of Bacteriophage, Cosmid, and Plasmid Clones
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • 0.7% top agarose (unit 1.1)
  • Host bacteria (OD 600 1.5 to 2 in 10 mM MgSO 4)
  • LB plates (unit 1.1)
  • Suspension medium (SM; unit 1.11)
  • Chloroform
  • Sterile round toothpicks (unit 1.1) or Pasteur pipet
  • Nitrocellulose membrane filters
  • Additional reagents and equipment for autoradiography ( appendix 3A) and phage titering (unit 1.11)
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Figures

Videos

Literature Cited

Literature Cited
   Kaiser, K. and Murray, N.E. 1984. The use of phage lambda replacement vectors in the construction of representative genomic DNA libraries. In DNA Cloning: A Practical Approach, Vol. 1 (D.M. Glover ed.) pp. 1‐47. IRL Press, Oxford.
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