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Related Experiment Videos

Automated template quantification for DNA sequencing facilities.

Kathryn M Ivanetich1, Wilson Yan, Kathleen M Wunderlich

  • 1Biomolecular Resource Center, University of California San Francisco, Surge 104, 90 Medical Center Way, San Francisco, CA 94143-0541, USA. kathyi@cgl.ucsf.edu

Journal of Biomolecular Techniques : JBT
|February 8, 2006
PubMed
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Automated PicoGreen assay quantifies plasmid DNA, improving DNA sequencing quality. This method is fast, cost-effective, and suitable for diverse double-stranded DNA templates in core labs.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Laboratory Automation

Background:

  • Accurate quantification of plasmid DNA is crucial for downstream applications like DNA sequencing.
  • Manual quantification methods can be time-consuming and prone to variability.
  • The PicoGreen dye assay offers sensitive DNA detection but requires efficient automation for high-throughput labs.

Purpose of the Study:

  • To automate the PicoGreen dye binding assay for plasmid DNA quantification.
  • To assess the impact of automated quantification on DNA sequencing quality in a core laboratory setting.
  • To evaluate the efficiency, accuracy, and cost-effectiveness of the developed protocol.

Main Methods:

  • Automation of pipetting, mixing, and reading of standards, blanks, and unknowns using the PicoGreen assay.

Related Experiment Videos

  • Generation of standard curves using pUC19 DNA across a defined concentration range.
  • Quantification of over 35,000 user-submitted plasmid DNA templates and assessment of sequencing data.

    • Main Results:

    • The automated protocol demonstrated high precision with coefficients of variance of 0.1 and low percent errors (1-7%) for pUC19 replicates.
    • Standard curves were nonlinear within the required assay range, necessitating careful concentration calculations.
    • 87% of user-submitted plasmids deviated by >=20% from the target concentration, highlighting the need for accurate quantification.
    • Automated quantification significantly improved DNA sequencing quality, as evidenced by data from 418 sequencing reactions.

    Conclusions:

    • The automated PicoGreen assay provides a robust, accurate, and efficient method for plasmid DNA quantification in core laboratories.
    • Implementing this automated protocol enhances DNA sequencing quality by ensuring accurate template concentrations.
    • The protocol is versatile, cost-effective (approx. $0.20/unknown), time-saving (1 hr/user), and adaptable for various double-stranded DNA samples.