DNA-quantification using Varioskan® and PicoGreen® dsDNA Quantitation Kit

Abstract

PicoGreen dsDNA Quantitation Reagent (Molecular Probes) is a sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for subcloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays¹.

The most commonly used technique for measuring nucleic acid concentration is the determination of absorbance at 260 nm (A260). The major disadvantages of the absorbance method are the large relative contribution of nucleotides, single-stranded nucleic acids and proteins to the signal, the interference caused by contaminants commonly found in nucleic acid preparations, the inability to distinguish between DNA and RNA, and the relative insensitivity of the assay (an A260 of 0.1 corresponds to a 5 ìg/mL dsDNA solution).

The combination of the Varioskan and PicoGreen kit is ideal for quantification of dsDNA. In a 96 well plate, a maximum of 88 preps can be analyzed, 8 wells are necessary for the standard curve. To simplify the assay procedure, the diluted PicoGreen reagent is dispensed with Varioskan in-build dispenser.