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Sequencing DNA Fragments Cloned into M13 and Phagemid Vectors

  • Neil Brewis
Protocol
  • 100 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The dideoxy chain-termination method (1) involves enzymatic elongation of a oligonucleotide primer that is annealed to a single-stranded DNA template. Single-stranded DNA of bacteriophage M13 (2) or phagemid vectors (3) give the most consistently satisfactory sequencing data. However, denatured double-stranded plasmid DNA can also serve as a suitable template and can also yield several hundred nucleotides of sequence information per reaction.

Keywords

Microfuge Tube Potassium Acetate Phagemid Vector Enzymatic Elongation VCSM13 Helper Phage 
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.
    Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.PubMedCrossRefGoogle Scholar
  2. 2.
    Messing, J. (1993) M13 cloning vehicles: their contribution to DNA sequencing, in DNA Sequencing Protocols (Griffin, H. G. and Griffin, A. M., eds.), Humana Press, Totowa, NJ, pp. 9–22.CrossRefGoogle Scholar
  3. 3.
    Zagursky, R. J. and Berman, M. L. (1984) Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene 27, 183–191.PubMedCrossRefGoogle Scholar
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    Gerischer, U. and Diirre, P. (1993) Sequencing using custom designed oligonucleotides, in DNA Sequencing Protocols (Griffin, H. G. and Griffin, A. M., eds.), Humana Press, Totowa, NJ, pp. 75–82.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Neil Brewis
    • 1
  1. 1.Marie Curie Research InstituteOxfordUK

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