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Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye
Published on: November 5, 2010
Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye
Johanna Leggate1, Ray Allain, Leah Isaac
1Ottawa Laboratory (Carling), Canadian Food Inspection Agency, Bldg. No. 22, CEF, Ottawa, Ontario, Canada, K1A 0C6.
Abstract:
A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25-2,500 pg/microl), and was not affected by a variety of contaminants in the assay mixture.
