Preparation of templates for DNA sequencing.

B. E. Slatko, P. Heinrich, B. Tracy Nixon, R. L. Eckert

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 lambda-derived phage as a source of DNA, a protocol for preparing lambda 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.

Original languageEnglish (US)
JournalCurrent protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
VolumeChapter 7
StatePublished - Jan 1 2001

All Science Journal Classification (ASJC) codes

  • Molecular Biology

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