Automated template quantification for DNA sequencing facilities.
The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.
Ivanetich
Kathryn M
KM
Biomolecular Resource Center, University of California San Francisco, Surge 104, 90 Medical Center Way, San Francisco, CA 94143-0541, USA. kathyi@cgl.ucsf.edu
Yan
Wilson
W
Wunderlich
Kathleen M
KM
Weston
Jennifer
J
Walkup
Ward G
WG
Simeon
Christian
C
D010957:Plasmids; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic