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PubMed · 16461949

Automated template quantification for DNA sequencing facilities.

Kathryn M Ivanetich·Wilson Yan·Kathleen M Wunderlich·Jennifer Weston·Ward G Walkup·Christian Simeon
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Abstract

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.

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PlasmidsSequence Analysis, DNATemplates, GeneticJournal ArticleResearch Support, N.I.H., ExtramuralValidation Study

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Kathryn M Ivanetich, Wilson Yan, Kathleen M Wunderlich, Jennifer Weston, Ward G Walkup, Christian Simeon. 2005. Automated template quantification for DNA sequencing facilities.. https://pubmed.ncbi.nlm.nih.gov/16461949/

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