Production of a Genomic DNA Library

Thomas Quertermous1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.7
DOI:  10.1002/0471142727.mb0507s13
Online Posting Date:  May, 2001
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Abstract

The describes the strategy of producing a genomic DNA library. A number of small‐scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (λ and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large‐scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.

     
 
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Table of Contents

  • Section IV: Production of Genomic DNA and cDNA Libraries
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Vector DNA (phage arms, cosmid arms, or linearized plasmid)
  • Insert fragment (units 5.3 5.6)
  • 10× DNA ligase buffer (unit 3.4)
  • T4 DNA ligase (measured in cohesive‐end units; New England Biolabs; unit 3.14)
  • Packaging extract (phage and cosmid) or competent E. coli (plasmid)
  • LB or LB/ampicillin plates (unit 1.1)
  • Top agarose containing 10 mM MgSO 4 (unit 1.1)
  • Additional reagents and equipment for plating, packaging, and titering bacteriophage (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, Vol. 1: A Practical Approach (D. Glover, ed.) pp. 1‐47. IRL Press, Oxford.
   Rodriguez, R.L. and Tait, R.C. 1983. Recombinant Techniques: An Introduction. Addison‐Wesley, Reading, MA.
   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.
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