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Production of Double-Stranded cDNA for Gene Library Synthesis

  • Jane Kirk
  • Steve Mayall
Protocol
  • 119 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The synthesis of complementary DNA (cDNA) from an mRNA template by the action of reverse transcriptase is a fundamental technique in molecular cloning. The objective is to make a large number of long cDNA copies. The quality and length of the cDNA product largely depends on the quality of the mRNA used as the starting material. Great care must therefore be taken to avoid mRNA degradation. The choice of primers and RTase used during the synthesis also influences the final product.

Keywords

Avian Myeloblastosis Virus cDNA Synthesis Reaction Avian Myeloblastosis Virus Reverse Transcriptase Synthesis Buffer Radioactive Nucleotide 
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

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    Gubler, U. and Hoffman, B. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269.PubMedCrossRefGoogle Scholar
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    Okayama, H. and Berg, P. (1982) High-efficiency cloning of full-length cDNA. Mol Cell. Biol 2, 161–170.PubMedGoogle Scholar
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    Elledge, S. J., Mulligan, J. T., Ramer, S. W., Spottswood, M., and Davis, R. W. (1991) λYES: A multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl. Acad. Sci. USA 88, 1731–1735.PubMedCrossRefGoogle Scholar
  4. 4.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Jane Kirk
    • 1
  • Steve Mayall
    • 1
  1. 1.ICRF, Clare Hall LaboratoriesSouth MimmsUK

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