Unit

UNIT 7.3 Preparation of Templates for DNA Sequencing

  1. Barton E. Slatko (λ templates, alkali denaturation, thermal cycle sequencing templates)1,
  2. Peter Heinrich (double-stranded templates)2,
  3. B. Tracy Nixon (single-stranded templates)3,
  4. Richard L. Eckert (pSP64CS/pSP65CS)4

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0703s21

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Slatko, B. E., Heinrich, P., Nixon, B. T. and Eckert, R. L. 2001. Preparation of Templates for DNA Sequencing. Current Protocols in Molecular Biology. 21:7.3:7.3.1–7.3.10.

Author Information

  1. 1

    New England Biolabs, Beverly, Massachusetts

  2. 2

    Consortium für Elektrochemische Industrie, Munich, Federal Republic of Germany

  3. 3

    Pennsylvania State University, University Park, Pennsylvania

  4. 4

    Case Western Reserve University, Cleveland, Ohio

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JAN 1993

Abstract

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a λ-derived phage as a source of DNA, a protocol for preparing λ phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double-stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.