Plating and Transferring Bacteriophage Libraries

Thomas Quertermous1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.1
DOI:  10.1002/0471142727.mb0601s34
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The usefulness of a recombinant phage library depends on the ability to screen a large number of phage and identify the clone that carries the DNA sequence of interest. This unit presents a protocol in which phage are allowed to multiply in host bacteria in a thin layer of agarose on regular bacterial plates. When nitrocellulose is applied to the agarose, phage particles and unpackaged DNA adsorb to the filter to produce a replica of the plate surface. If the agarose surface is not excessively wet, there will be little spreading of the phage on the filter. Subsequent treatment of the filter with sodium hydroxide destroys the phage particles and denatures the phage DNA which then binds to the nitrocellulose. Neutralization of the filters is required to maintain the integrity of the nitrocellulose. Hybridization of these filters to a DNA or RNA probe will identify the location of the phage plaque of interest, which can then be recovered from the plate.

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

Table of Contents

  • Section I: Plating Libraries and Transfer to Filter Membranes
  • Commentary
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • Host bacteria, selection strain if applicable (unit 1.10; Table 97.80.4711; Table 97.80.4711)
  • Recombinant phage (unit 5.10)
  • 0.7% top agarose (prewarmed; unit 1.1)
  • 82‐mm or 150‐mm LB plates; or 245 × 245–mm Nunc bioassay LB plates (unit 1.1)
  • 0.2 M NaOH/ 1.5 M NaCl
  • 0.4 M Tris⋅Cl, pH 7.6/ 2× SSC
  • 2× SSC ( appendix 22)
  • Nitrocellulose membrane filters (or equivalent)
  • 20‐G needle
  • 46 × 57–cm Whatman 3MM or equivalent filter paper
  • 80°C vacuum oven or 42°C oven
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Arber, W. 1983. A beginner's guide to lambda biology. In Lambda II (R.W. Hendrix, J.W. Roberts, F.W. Stahl, and R.A. Weisberg eds.) pp. 381‐395. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
   Arber, W., Enquist, L., Hohn, B., Murray, N. and Murray, K. 1983. Experimental methods for use with lambda. In Lambda II (R.W. Hendrix, J.W. Roberts, F.W. Stahl, and R.A. Weisberg eds.) pp. 433‐466. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
   Williams, B.G. and Blattner, F.R. 1980. Bacteriophage lambda vectors for DNA cloning. In Genetic Engineering, Vol. 2 (J.K. Setlow and A. Mullander eds.) p. 201. Plenum NY.
Key References
   Benton, W.D. and Davis, R.W. 1977. Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196:180‐182.
  Describes the method of plaque hybridization developed by the authors to allow isolation of phage possessing specific cloned DNA sequences.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library