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Nucleic acids are isolated from sample material such as cells and subsequently employed in further laboratory experiments. For this purpose, it is advisable to determine the concentration of the purified DNA or RNA as well as verify their purity. In this way, accurately defined amounts of the nucleic acid will enter the downstream application, and any contaminations that may impact a sensitive reaction or assay are detected in real time.
UV-Vis spectrophotometry is an easy, quick and time-tested method to achieve these objectives. In the range of 260 nm, nucleic acids show a characteristic absorbance peak (figure 1). This absorbance value is therefore used to calculate nucleic acid concentrations. According to the Lambert-Beer law, two parameters are required for the calculation of the concentration of a sample: the optical path length (= light path length (L)) and the molar extinction coefficient (material and wavelength-specific constant) of the sample to be measured. The specific factor (F) can be calculated when using a standard cuvette with a light path of 1 cm. This factor is then multiplied by the measured absorbance value (A) in order to arrive at the concentration (C) of the sample solution. Molar extinction coefficients and specific factors, respectively, are typically available in the literature. The factor for dsDNA, for example, is 50 µg/mL (RNA: 40 µg/mL), and it is by definition equivalent to one absorbance unit. these data.
|Lambert-Beer law: |
C = A/(L x Ɛ )
F = 1/Ɛ (for a light path of 1 cm)
- C = A x F
| C = Concentration |
A = Absorbance
L = Optical (light) path length
Ɛ = Molar extinction coefficient
(sample and wavelength-specific)
F = Factor