DNA quantitation and normalization are critical steps for a wide range of
molecular biology applications such as cloning, quantitative PCR (qPCR) and
next-generation sequencing (NGS). Fluorescent dye-based DNA quantitation
uses specially designed DNA binding compounds that intercalate only with
double-stranded DNA (dsDNA) molecules. When excited by a specific
wavelength of light, only dye in the DNA-bound state will fluoresce. These
aspects of the technique contribute to low background signal, high accuracy
and specificity—making it a suitable measure of DNA concentration prior to
advanced downstream applications.
Another desirable attribute of fluorescence quantitation over other methods is
ease of use and scalability for laboratories conducting all levels of throughput.
In this application note, we demonstrate the flexibility of the QuantiFluor®
dsDNA System for use in 40μl reaction volumes compatible with 384-well
plates. We also provide guidance for optimization of the assay according to
variables such as dynamic range, sensitivity, accuracy, cost per reaction and
other features of high-throughput workflows.