How is concentration calculated from A260?

It uses the Beer-Lambert law at 260 nanometres. Concentration in ng per uL equals the A260 reading times an extinction factor times the dilution factor. The factor is the ng per uL that gives an absorbance of 1.0 in a 1 centimetre path.

Why do DNA and RNA use different factors?

The factor reflects how strongly each nucleic acid absorbs at 260 nm. Double-stranded DNA uses 50, single-stranded RNA uses 40, and single-stranded DNA and short oligonucleotides use about 33. Using the wrong factor scales your result incorrectly.

What does the A260/A280 ratio tell me?

It flags protein or phenol contamination, both of which absorb at 280 nm. Pure DNA reads about 1.8 and pure RNA about 2.0. A ratio well below those values means your sample is contaminated and may inhibit downstream enzymes.

What does the A260/A230 ratio tell me?

It flags contamination by salts, EDTA, carbohydrates, and phenol, which absorb near 230 nm. A clean sample sits around 2.0 to 2.2. A low A260/A230 ratio is common after column purification and often signals residual guanidine or wash buffer.

Does the path length matter?

Yes. The extinction factors assume a 1 centimetre path length, which is the standard for both cuvette and microvolume spectrophotometers that normalise to 1 cm. If your instrument uses a different path and does not normalise, scale the result accordingly.

DNA/RNA Quantitation by Absorbance (A260) Calculator

Measuring nucleic acid concentration by ultraviolet absorbance is the fastest routine quantification in molecular biology. A single reading at 260 nanometres, the wavelength where the bases absorb most strongly, converts directly into a concentration, and two extra readings tell you how clean the sample is.

How it works

The Beer-Lambert law makes absorbance proportional to concentration. Rearranged for nucleic acids:

concentration (ng/uL) = A260 x factor x dilution

The factor is the concentration that produces an absorbance of exactly 1.0 in a 1 cm path. It differs by molecule because each absorbs differently at 260 nm:

dsDNA  -> 50
ssRNA  -> 40
ssDNA  -> 33
oligo  -> 33 (single-stranded; sequence-dependent)

Multiplying by the dilution factor recovers the concentration of the original undiluted stock.

Purity ratios and notes

Two ratios reveal contamination. The A260/A280 ratio detects protein and phenol, which absorb at 280 nm; pure DNA reads about 1.8 and pure RNA about 2.0. The A260/A230 ratio detects salts, EDTA, carbohydrate, and phenol, which absorb near 230 nm; a clean sample reads about 2.0 to 2.2. Ratios well below these targets mean the preparation may inhibit PCR, restriction digests, or sequencing.

The extinction factors assume a 1 cm path length, the standard for cuvette and microvolume instruments. The oligonucleotide factor is a general default; for an exact answer on a short oligo, compute the extinction coefficient from its sequence.