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 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.
Thompson
Mark
M
College of Science and Management, University of Northern British Columbia, Prince George, British Columbia, Canada.
Aukema
Kelly
K
O'Bryan
Dana
D
Rader
Stephen
S
Murray
Brent
B
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D013010:Sonication / Q000295:instrumentation; D013698:Templates, Genetic
D004278:DNA, Superhelical; D004247:DNA