Reading DNA and RNA concentration from A260

Nucleic acids absorb ultraviolet light at 260 nm, and how much they absorb is proportional to how much is there. A spectrophotometer or NanoDrop reports that absorbance as A260, and a single factor turns it into a concentration.

The factor

At A260 = 1.0 over a 1 cm path, a solution holds a fixed mass concentration that depends on the molecule:

Double-stranded DNA: 50 µg/mL
Single-stranded DNA: 33 µg/mL
RNA: 40 µg/mL

Single-stranded molecules absorb more per unit mass because base stacking, which suppresses absorbance, is weaker — so a lower mass gives the same A260.

The calculation

concentration (ng/µL) = A260 × factor × dilution factor

A dsDNA sample read at A260 = 0.5 after a 10× dilution: 0.5 × 50 × 10 = 250 ng/µL. Note ng/µL and µg/mL are numerically identical, so the same number works in both units. Don’t forget the dilution — reading a diluted sample without multiplying back is the most common mistake.

Purity, separately

A260 gives quantity, not purity. The A260/A280 ratio checks for protein or phenol contamination: pure DNA reads ~1.8 and pure RNA ~2.0. A low ratio means something else is absorbing at 260 nm too, which inflates your concentration estimate. A260/A230 catches guanidinium and other carryover.

When A260 isn’t enough

Absorbance can’t tell target DNA from contaminating nucleic acids or free nucleotides, and it is unreliable at very low concentrations. For sensitive or fragment-specific quantification — such as library QC — a fluorometric assay (Qubit) or a fragment analyzer is more accurate.