Nucleic Acid Quantification Calculator

Why A260 gives a concentration

The aromatic rings in nucleotide bases absorb ultraviolet light with a peak near 260 nm. Beer’s law makes that absorbance proportional to how much nucleic acid the beam passes through, so a spectrophotometer reading converts straight into a concentration once you know the proportionality constant. That constant is the extinction factor: the concentration in µg/mL that would give an absorbance of exactly 1.0 in a 1 cm path.

The factor depends on what you have, because base stacking in a double helix suppresses absorbance (hypochromism); melting the helix removes it, which is the hyperchromic effect. Double-stranded DNA takes 50, single-stranded DNA 33, RNA 40 and short oligonucleotides about 33. Multiply by your dilution factor and you have the concentration of the original stock. Because 1 µg/mL and 1 ng/µL are the same number, no scaling is needed between the two units people quote.

Worked example

A genomic DNA prep is diluted 1 in 20 and reads A₂₆₀ = 0.184 against a buffer blank.

• c = A₂₆₀ × factor × dilution
• c = 0.184 × 50 × 20
• c = 184 ng/µL, equivalently 184 µg/mL

If the whole prep is 50 µL, that is 9.2 µg of DNA in total. To load 500 ng into a reaction you would take 2.72 µL. Both figures appear in the readouts, because those are the two numbers you actually need at the bench and both are easy to fumble at 9 pm.

Purity ratios, and what absorbance cannot see

The concentration is only meaningful if the absorbance is coming from your nucleic acid. Two ratios check that. A260/A280 near 1.8 indicates reasonably clean DNA and near 2.0 reasonably clean RNA; a lower value means protein or phenol, both of which absorb strongly at 280 nm. A260/A230 below about 2.0 points to guanidine, EDTA or carbohydrate carried over from the extraction.

What absorbance fundamentally cannot do is tell one nucleic acid from another. Free nucleotides, degraded fragments, residual RNA in a DNA prep and your intact target all absorb at 260 nm, so a UV reading is a total rather than a specific measurement. That distinction matters for library prep, and for quantifying DNA in an RNA background. In those cases use a dye-based fluorometric assay, which only lights up when bound to its intended target.

Common mistakes

• Using 50 for RNA. RNA’s factor is 40, so applying the dsDNA factor overstates the concentration by 25 percent. This is the most frequent error in this calculation and it propagates into every downstream reaction.
• Reading outside the linear range. Keep A₂₆₀ between about 0.1 and 1.0. Below 0.05 the reading is mostly instrument noise; above 1.0 too little light reaches the detector and the response stops being linear. Dilute and re-read rather than trusting a high number.
• Blanking against the wrong solution. Blank with the exact buffer the sample is in. Blanking water against a TE-suspended sample leaves a baseline offset that hits low-concentration samples hardest.
• Forgetting the dilution factor. A tenfold dilution left at 1 in the calculator understates the stock by a factor of ten. Enter 1 only when you read the sample neat.