Which materials are required for this method?
Quantification of nucleic acids and proteins benefits from the selection of fluorescent dyes that bind to their respective target molecule with high specificity. In addition, at least one standard of known concentration must be available. Excitation of the fluorophore and measurement of the fluorescence intensity of the emitted light require either a stand-alone fluorometer or a fluorescence module that is integrated within another instrument. Equipment and fluorescent dye must be compatible so that the light source inside the instrument provides the required excitation wavelength(s) and the detector measures the light emitted. Depending on the type of instrument, the reagent solutions are located either in thin-walled reaction vessels or in cuvettes. Compatibility with the specific application with respect to fit, transparency, auto-fluorescence and volume must be taken into consideration.
How is the method carried out?
Figure 2 shows the sequence of a fluorometric measurement. Preparation of the blank, the standards and the sample is initiated by the addition of the fluorescent dye, followed by a short incubation period. First, one or more standards of known concentrations are measured in order to generate the standard curve. Determination of sample concentration subsequently takes place in a second step, where the measured fluorescence intensities of the samples are evaluated in relation to the standard curve.