Improved Purification of the Double-Stranded RNA from Killer Strains of Yeast

plying by its reciprocal). To use that value for microplate samples, absorbance values must be normalized to a 1 cm path length. This can be done by determining a conversion factor for a given sample volume and microplate. Alternatively, several microplate spectrophotometers use infrared absorbance measurements to determine path length on a well-by-well basis, and absorbance values can be normalized automatically. The A-1 value for double-stranded DNA at 260 nm is commonly approximated as 50 cm μg/mL for a 1 cm path length (2,5). The dependence of nucleic acid absorbance on ionic strength has been reported previously (1,6), but is not generally recognized. Figure 3 illustrates the difference in absorbance of DNA prepared in water, Tris (TE) buffer and Tris-buffered saline (TES). A260 values were approximately 15% lower in TE and 23% lower in TES when compared to the same concentrations in water. The calculated A-1 values are 38, 45 and 50 cm μg/mL for DNA in water, TE and TES, respectively (Figure 3). Thus, the commonly used value of 50 is correct only for relatively high ionic strength solutions. A lower detection limit for DNA by A260 measurement may be achieved if the samples are dissolved in deionized water rather than a salt solution. With attention to technique, accurate and reproducible DNA measurements can be made easily in microplates. The lower limits of detection and quantitation are comparable to those obtained with semi-micro cuvettes in conventional spectrophotometers.