Plating and Transferring Cosmid and Plasmid Libraries

John H. Weis1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.2
DOI:  10.1002/0471142727.mb0602s24
Online Posting Date:  May, 2001
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Abstract

There are two commonly used protocols for the screening of recombinant bacteria with hybridization probes. One method involves the spreading of bacteria on the surface of agar using a sterile spreader. A nitrocellulose membrane filter is then placed on top of the colonies and most of each colony is transferred to the filter. This method works well when relatively small numbers of positive colonies are being selected (up to several thousand). A second method, described in this unit, employs a matrix of some type (here nitrocellulose filters are used) upon which bacteria can be plated and grown when the filter is placed on top of a nutrient agar surface. Once the plated bacteria have grown into visible colonies, the filters can be used for replica plating and in situ hybridization analysis.

     
 
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Table of Contents

  • Commentary
  • Key Reference
     
 
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Materials

Basic Protocol 1:

  Materials
  • LB plates containing antibiotic (unit 1.1)
  • LB medium (unit 1.1)
  • LB plates containing 50 µg/ml chloramphenicol (unit 1.1)
  • 0.5 M NaOH
  • 1 M Tris⋅Cl, pH 7.5
  • 0.5 M Tris⋅Cl, pH 7.5/ 1.25 M NaCl
  • 10‐ or 15‐cm Whatman 3MM or equivalent filter paper discs
  • Sintered glass filter with vacuum
  • Nitrocellulose membrane filters (10‐ or 15‐cm, Millipore HATF)
  • 20 × 20–cm Whatman 3MM or equivalent filter paper
  • 20 × 20–cm glass plate
  • 20‐G needle
  • 46 × 57–cm Whatman 3MM or equivalent filter paper
  • 80°C vacuum oven
  • NOTE: All materials coming into contact with E. coli must be sterile.
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Figures

Videos

Literature Cited

Key Reference
   Hanahan, D. and Meselson, M. 1983. Plasmid screening at high density. Meth. Enzymol. 100:333‐342.
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