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Amplification of a Bacteriophage Library

Lloyd B. Klickstein1

1Brigham and Women's Hospital, Boston, Massachusetts

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 5.10
DOI: 
10.1002/0471142727.mb0510s34
Online Posting Date: 
May, 2001
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Abstract

This protocol may be used for genomic DNA or cDNA libraries. A freshly packaged and titered library is adsorbed to log phase plating bacteria. The mixture is then plated at high density and allowed to grow until the plaques are just subconfluent. The phage are eluted from the plate by overnight incubation with phage buffer and the library is titered and stored.

     
 
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Table of Contents

  • Section V: Amplification of Transformed or Packaged Libraries
  • Basic Protocol
  • Commentary
  • Tables
     
 
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Materials

 Basic Protocol
 Materials
  • LB medium containing 0.2% maltose and 10 mM MgSO4 (UNIT 1.1)
  • Suitable host (Table 5.10.1)
  • In vitro packaged phage library (UNITS 5.7 & 5.8A)
  • Top agarose (UNIT 1.1),warmed to 47°C
  • 150-mm H plates (UNIT 1.1), warmed to 37°C
  • Suspension medium (SM; UNIT 1.11)
  • Chloroform
  • Dimethyl sulfoxide (DMSO)
  • Additional reagents and equipment for titering bacteriophage (UNIT 1.11)
     
    Table 5.10.1 Suitable Escherichia coli Host Strains for Amplifying Lambda-Constructed Libraries
    VectorE. coli hostRelevant host genotype
    gt10C600hflAhflA
    gt11Y1088SupF, lacIq (no anti-biotics needed)
    EMBL 3 or 4P2392, Q359, NM539P2 lysogen
    Charon 4ALE392SupF
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