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DNA Sequencing of Plasmids

  • George Murphy
Protocol
  • 94 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

In double-stranded DNA sequencing, the two DNA strands must first be separated to enable the primer to bind to the priming site. This may be done by treating the DNA with alkali. Conventionally, use of the alkali denaturation method involves neutralization of the sample by acid treatment followed by ethanol precipitation and recovery of the DNA by centrifugation. The time-consuming process of ethanol precipitation may be avoided by neutralizing the alkali-treated DNA and recovering the sample in its original volume by passage through a spin-dialysis column. The use of spin-dialysis confers the additional advantage of cleaning up the template by removing traces of low-molecular-weight compounds that may interfere with the sequencing reactions. The method used here to prepare plasmid is a variation on the boiled-lysis method (1), using a rapid approach to avoid phenol extraction.

Keywords

Sodium Hydroxide Fresh Tube Plasmid Preparation Blue Dextran Lysozyme Solution 
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.

Reference

  1. 1.
    Holmes, D. S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193–197.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • George Murphy
    • 1
  1. 1.John Innis Centre for Plant Science ResearchNorwichUK

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