Spectrophotometric quantification of DNA and RNA

This post was written by admin on June 2, 2009
Posted Under: General

Because DNA and RNA absorb ultraviolet light, with a absorption peak at 260nm wavelength, spectrophotometers are commonly used to determine the concentration of DNA in a solution. Inside a spectrophotometer, a sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample.

Using the Beer-Lambert law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule. At a wavelength of 260 nm, the extinction coefficient for double-stranded DNA is 50 (μg/ml)-1 cm-1; for single-stranded DNA and RNA it is 38 (μg/ml)-1 cm-1. Thus, an optical density (or “OD”) of 1 corresponds to 50 µg/ml for double-stranded DNA, 38 µg/ml for single-stranded DNA and RNA. This method of calculation is valid for up to an OD of at least 2

Concentration of double stranded DNA = absorvance at 260nm * 50 * dilution (if you diluted 1 in 100 to quantify, the dilution value in this case will be 100)

Concentration of single-stranded DNA and RNA = absorvance at 260nm * 38 * dilution (if you diluted 1 in 50 to quantify, the dilution value in this case will be 50)

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