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Preparing Libraries in Escherichia coli

  • Alexander V. Tobias
Part of the Methods in Molecular Biology™ book series (MIMB, volume 231)

Abstract

The process of preparing libraries of mutagenized or recombined gene sequences for screening or selection in Escherichia coli is a special application of cohesive-end subcloning (1). PCR products are digested with restriction endonucleases, ligated into an expression vector digested with the same enzymes, and the resultant recombinant plasmids are transformed into supercompetent bacteria. One difference between routine subcloning of a single gene and preparing libraries is that in the latter case, there can be little allowance for the presence of transformants containing recircularized vector and no insert (so-called “background” ligation products). Furthermore, ligation and transformation of the recombinant plasmids must be performed using materials and conditions that yield a sufficient number of transformants (∼103–105) for identifying variants exhibiting desired properties.

Keywords

Recombinant Plasmid Transformation Efficiency Zymo Research Ligation Reaction Ligation Product 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Delidow, B. C. (1993) Molecular Cloning of Polymerase Chain Reaction Fragments with Cohesive Ends, in PCR Protocols—Current Methods and Applications (White, B. A., ed.), Humana Press, Totowa, NJ, pp. 217–228.Google Scholar
  2. 2.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  3. 3.
    Conley, E. C. and Saunders, J. R. (1984) Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol. Gen. Genet. 194, 211–218.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Alexander V. Tobias
    • 1
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadena

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