Application of the DNA-specific dye EvaGreen for the routine quantification of DNA in microplates

For the quantiWcation of DNA, most laboratories rely on the direct determination of the optical absorption of DNA at 260 nm [1]. This method is insensitive, susceptible to interference, and diYcult to be carried out automatically. For the quantiWcation of proteins, similar techniques have long since been superseded by indirect methods based on dye incorporation or chemical modiWcations [2]. The Xuorescent dye PicoGreen has been developed for the quantiWcation of DNA by Xuorescence spectroscopy [3,4]. Here we employed the DNA-speciWc Xuorescent dye EvaGreen (Biotium, www.biotium.com) for the setup of a costeYcient alternative dye association assay for the routine quantiWcation of DNA. This dye originally was intended for use in quantitative PCR. EvaGreen–DNA complexes exhibit an intense Xuorescence with an excitation maximum at 503 nm and a broad green emission with a maximum at 527 nm. For establishing the detection range of this dye, commercial DNA standards of known concentration were diluted to 95 l with assay buVer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid [EDTA],1 20 mM NaCl, pH 8.0) supplemented with 5 l of a 20-fold concentrated stock of EvaGreen and incubated at 72 °C for 5 min. Subsequently, the Xuorescence emission was recorded with a Xuorescence spectrometer equipped with 3 £ 3-mm cuvettes. The observable signals displayed a DNA concentration-dependent increase that saturated at approximately 0.5 g DNA (Fig. 1A). The Xuorescence signal was intensive and susceptible to fading, eVectively requiring a signiWcant reduction in the intensity of the excitation