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.

What is the DNA/RNA Quantitation by Absorbance (A260) Calculator?

Apply the correct A260 extinction factor (50 for dsDNA, 40 for ssRNA, 33 for ssDNA and oligos) with your dilution factor to compute ng/uL, and check the A260/A280 and A260/A230 purity ratios. For every molecular biology lab. It runs free in your browser on Gera Tools, with nothing uploaded.

DNA/RNA Quantitation by Absorbance (A260) Calculator

Name: DNA/RNA Quantitation by Absorbance (A260) Calculator
Creator: Gera Tools
License: https://creativecommons.org/licenses/by/4.0/

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.

Practical lab guidance

When to trust your A260 reading — and when to be skeptical

Spectrophotometric quantification is fast but has blind spots. The Beer-Lambert law assumes that absorbance at 260 nm comes entirely from nucleic acid bases. Several contaminants also absorb at or near 260 nm — free nucleotides, RNA degradation products, and some organic solvents — which means a contaminated sample can read as higher concentration than it actually is. The purity ratios are the first check, but they have limits too: a sample can have acceptable A260/A280 and A260/A230 ratios while still containing non-UV-absorbing inhibitors such as polysaccharides, EDTA, or detergents.

For sensitive downstream applications (next-generation sequencing library preparation, qPCR, single-cell work), it is common practice to supplement spectrophotometric measurement with a fluorescence-based method such as a Qubit assay, which uses a dye that binds specifically to dsDNA and is largely unaffected by RNA, protein, or oligonucleotides. The spectrophotometric result gives total nucleic acid; the fluorescence result gives the specific target molecule.

Choosing the right extinction factor

The four factors in this tool (50, 40, 33, 33) are the standard rules of thumb:

50 for dsDNA applies to double-stranded genomic DNA and linear PCR products. The hypochromicity of the double helix — base stacking reducing absorbance — is baked into this value.
40 for ssRNA applies to single-stranded RNA such as total RNA preparations, mRNA, or in vitro transcripts. RNA absorbs slightly less strongly than dsDNA per base, hence the lower factor.
33 for ssDNA and oligos applies to single-stranded DNA and synthesized oligonucleotides. For a short oligo, the true extinction coefficient depends on its exact sequence composition; 33 is a reasonable average but can introduce a few percent error for short, GC-rich or GC-poor sequences.

Interpreting purity ratios in practice

A260/A280 below 1.7 (DNA) or 1.9 (RNA): protein or phenol contamination. A re-clean step — column purification or phenol:chloroform extraction — is usually needed before sensitive enzyme-based applications.

A260/A230 below 1.8: common after column-based DNA purification, often from residual chaotropic salts (guanidinium) or ethanol wash carry-over. An additional wash step and complete drying of the column membrane before elution usually corrects this. Very low A260/A230 (below 1.5) can severely inhibit PCR.

Ratios above the “pure” range: occasionally seen when the A260 is very low and the absorbance values are near the instrument’s noise floor, making ratios unreliable. If your A260 is below about 0.05, dilute or re-elute and remeasure for a more reliable reading.

Dilution factor — when and why

Many spectrophotometers measure in pure solution without dilution (especially microvolume instruments like NanoDrop). If you measured a diluted aliquot instead, enter the dilution factor here to recover the concentration of the original undiluted stock. For example, if you diluted 1 µL into 49 µL (a 50× dilution) and measured the resulting solution, enter 50 as the dilution factor.