Abstract

Publisher Summary This chapter discusses the experimental methods needed to acquire a melting curve and the analysis and interpretation of the data. Any standard commercial UV spectrophotometer can be equipped to measure melting curves. A useful instrument is a single-beam Gilford (Oberlin, OH) spectrophotometer (Model 2530) with an automated reference compensator that allows melting curves to be obtained on three separate samples simultaneously. One major advantage of using UV spectroscopy is the high sensitivity of the method. Normally, the absorbance of the sample used should be between 0.2 and 2.0. Sample preparation for UV melting studies is straightforward. The RNA stock solution is prepared by dialysis against the desired buffer and different concentrations are made by dilution. The high salt concentration is chosen to minimize electrostatic repulsion between strands and to avoid divalent ions, which catalyze hydrolysis of RNA and favor triple-strand formation. This solvent provides a standard condition for measuring melting curves and for comparing results with previously published data.