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How do I determine the concentration, yield and purity of a DNA sample?

DNA yield can be assessed using four different methods: absorbance (optical density), agarose gel electrophoresis, fluorescent DNA-binding dyes and a luciferase-pyrophosphorylation-coupled quantitation system. While all of these methods are useful, each has caveats to consider when choosing a quantitation system. The most common technique to determine DNA yield and purity is also the easiest method—absorbance. All that is needed for measurement is a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes and a solution of purified DNA. Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. Below, we describe how to estimate concentration, yield and purity using absorbance. For more information about the other methods, see the DNA Purification chapter of the Protocols and Applications Guide.

DNA concentration can be estimated by measuring the absorbance at 260nm (A260), adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure DNA.

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml

Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (ml)

DNA purity can be estimated from the A260/A280 ratio. An A260/A280 ratio between 1.7 and 2.0 generally represents a high-quality DNA sample. The ratio can be calculated after correcting for turbidity (absorbance at 320nm).

DNA Purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)

Note: A spectrophotometer is most accurate when measurements are within the instrument's linear range (generally 0.1–1.0).