Plasmid sonication improves sequencing efficiency and quality in the Beckman Coulter CEQ system

We report on an unexpectedly high rate of unreadable chromatograms from plasmid sequencing using Beckman Coulter's protocols, chemistry, and CEQ8000 instrument. Failed or poor quality plasmid sequence chromatograms were accompanied by a sharp drop, fluctuation, or steady decline in the current...

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Bibliographic Details
Published in:	BioTechniques Vol. 45; no. 3; pp. 327 - 329
Main Authors:	Thompson, Mark, Aukema, Kelly, O'Bryan, Dana, Rader, Stephen, Murray, Brent
Format:	Journal Article
Language:	English
Published:	England Taylor & Francis Group 01-09-2008
Subjects:	Base Sequence DNA - genetics DNA, Superhelical - genetics Plasmids - genetics Sequence Analysis, DNA - instrumentation Sequence Analysis, DNA - methods Sonication - instrumentation Templates, Genetic
Online Access:	Get full text
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Summary:	We report on an unexpectedly high rate of unreadable chromatograms from plasmid sequencing using Beckman Coulter's protocols, chemistry, and CEQ8000 instrument. Failed or poor quality plasmid sequence chromatograms were accompanied by a sharp drop, fluctuation, or steady decline in the current and a corresponding delay in signal counts beyond the time of capillary injection. We observe a correlation between the presence of supercoiled DNA and these sequencing problems. Herein we demonstrate that plasmid sonication, which is known to fragment supercoiled DNA, is an effective way to improve sequence phred20 read lengths to the point that they are not significantly different from Beckman Coulter's control template or enzymatically linearized plasmids.
ISSN:	0736-6205 1940-9818
DOI:	10.2144/000112902