DNA Concentration Calculator — A260 to ng/µL

Background.

This calculator turns a UV absorbance reading at 260 nm into a nucleic acid concentration in ng/µL, a total yield in µg, the two purity ratios everyone quotes, and the pipetting volume that delivers whatever mass your next reaction needs. It is the arithmetic behind every spectrophotometer readout — written out so you can see which constant was applied and change it if your lab uses a different one.

The physics is Beer–Lambert: absorbance is proportional to concentration and to the distance light travels through the sample, A = ε·c·l. Rearranged for concentration, c = A ÷ (ε·l). Rather than carry a molar extinction coefficient, nucleic acid work uses a mass-based shortcut — a conversion factor in micrograms per millilitre per absorbance unit per centimetre. For double-stranded DNA that factor is 50, for single-stranded DNA 33, and for RNA 40. Those three numbers are the ones tabulated by Desjardins and Conklin in the Journal of Visualized Experiments, and they are what a NanoDrop-class instrument applies when you press the button. Double-stranded DNA has the lowest factor per microgram because base stacking inside an intact duplex suppresses absorbance — the hypochromic effect — so the same mass of DNA absorbs less when it is paired than when it is melted.

The conversion factor is the whole reason a page like this exists. It assumes a long, compositionally random polymer. It is a poor approximation for a 20-mer primer, for a homopolymer stretch, and for anything with unusual base composition, because the absorbance of a short oligonucleotide depends on which bases sit next to which. If you are quantifying an oligo, use a sequence-specific extinction coefficient from your supplier and enter it in the Custom field — do not use 33 and hope. Published tables also disagree with each other for single-stranded DNA: 33, 37 and 40 all appear in print, from different lineages of the same underlying measurement. This calculator uses 33 because it is the value the instrument that generated your reading almost certainly applied, and it exposes the field so you are not trapped in that choice.

The two ratios need more care than they usually get. A260/A280 is conventionally quoted as about 1.8 for pure DNA and about 2.0 for pure RNA, and A260/A230 as somewhere between 2.0 and 2.2. Those are conventions reported by instrument manufacturers, not specifications published by a standards body, and the same documents that publish them also report that moving the diluent from acidic to alkaline shifts A260/A280 by 0.2 to 0.3 in either direction. That artefact is wider than the band. Wilfinger, Mackey and Chomczynski measured the effect directly in 1997 and found RNA ratios moving from roughly 1.5 to 2.0 simply by taking the water from pH 5.4 to pH 7.5–8.5. So a ratio outside the band is a prompt to re-read the sample in 10 mM Tris·HCl at pH 8.0–8.5 before you conclude anything about purity. The calculator prints that caveat with every result rather than hiding it in an FAQ.

One more scope limit belongs up here rather than below the fold: absorbance measures how much material absorbs at 260 nm, and nothing else. It cannot tell an intact 20 kb genomic fragment from the same mass of sheared 200 bp debris, and it happily counts free nucleotides, degraded RNA and carry-over nucleic acid of the wrong type. This is why NIST assigns certified values to its human genomic DNA standard by PCR-based copy-number and mass-concentration methods rather than by absorbance, and why a fluorescent dye assay is the right tool when you need to know how much of a specific analyte is present in a mixed sample. Use A260 for a fast, non-destructive, reagent-free estimate of total nucleic acid. Use a dye-based assay when the number has to be right.

Everything below the widget — the derivation, the worked example, the ratio interpretation table and the FAQs — is generated from the same worked case the calculator computes, so the numbers in the prose and the numbers on screen cannot drift apart.

What is dna concentration calculator (a260)?

A260 quantification is the measurement of a nucleic acid solution's absorbance at 260 nanometres, the wavelength at which the purine and pyrimidine bases absorb most strongly, and its conversion into a mass concentration using an empirical factor. The relationship is the Beer–Lambert law: A = ε·c·l, where A is absorbance (dimensionless), ε is the extinction coefficient, c is concentration and l is the optical path length. Because ε for a nucleic acid depends on base composition and on whether the strands are paired, wet-lab practice replaces the molar coefficient with a mass-based conversion factor expressed in micrograms per millilitre per absorbance unit at a 1 cm path. Those factors — 50 for double-stranded DNA, 33 for single-stranded DNA, 40 for RNA — are averages over random sequence. An absorbance of 1.000 AU at 260 nm through a 1 cm path therefore means 50 ng/µL of double-stranded DNA, 33 ng/µL of single-stranded DNA, or 40 ng/µL of RNA. Two secondary readings are taken at the same time. A280 sits near the absorbance maximum of the aromatic amino acids tryptophan and tyrosine, so the A260/A280 ratio falls when protein is carried over. A230 sits where guanidine salts, EDTA, phenol and carbohydrates absorb, so the A260/A230 ratio falls when extraction reagents are carried over. Neither ratio is a concentration and neither is a specification: they are diagnostics whose value shifts measurably with the pH and ionic strength of the diluent, independently of how pure the sample actually is. Microvolume pedestal spectrophotometers complicate one detail worth knowing. They hold the sample in a liquid column typically 1 mm or 0.2 mm deep rather than in a 1 cm cuvette, then scale the raw absorbance to a 10 mm-equivalent value before reporting it. That is why you normally leave the path length in this calculator at 1 cm even when the physical path was a tenth of that — the instrument has already done the conversion.

How to use this calculator.

1. 01Choose the analyte. Double-stranded DNA (genomic preps, plasmid minipreps, PCR products, restriction fragments) uses 50. Single-stranded DNA uses 33. RNA — total RNA, mRNA, in vitro transcripts — uses 40. Pick Custom for a short oligonucleotide and enter the sequence-specific factor from your supplier's spec sheet.
2. 02Enter A260 exactly as the instrument reported it. If the reading is above about 1.0 AU or below about 0.1 AU, dilute or concentrate and read again rather than trusting an extrapolation outside the linear range.
3. 03Enter A280 and A230 if you measured them. Leave either at 0 if you did not — the calculator reports that ratio as 'not evaluated' rather than dividing by zero and returning nonsense.
4. 04Set the dilution factor to the fold dilution of the material that went into the cuvette. Reading neat is 1. Ten microlitres of sample plus ninety of buffer is 10. The concentration reported is always that of the original undiluted sample.
5. 05Leave the path length at 1 cm for a standard cuvette and for microvolume pedestal instruments, which already normalise to a 10 mm equivalent. Change it only if your instrument reports raw absorbance at a physical short path.
6. 06Enter your elution volume as the sample volume to get the total yield of the whole prep in micrograms.
7. 07Enter the mass your next reaction needs — a ligation, a sequencing submission, a library prep — and the calculator returns the microlitres of this sample that deliver it.
8. 08Read the ratios as flags, not grades. If either is outside its band, re-read the sample in 10 mM Tris·HCl at pH 8.0–8.5 before you re-purify anything: the pH artefact alone is bigger than the band.

The formula.

Four equations run this page, and all of them are one rearrangement away from Beer–Lambert.

c[µg/mL] = (A260 / l) × F × D concentration of the original sample Y[µg] = c[ng/µL] × V[µL] / 1000 total yield of the whole prep R280 = A260 / A280 protein-carryover ratio R230 = A260 / A230 reagent-carryover ratio Vneed = m_target[ng] / c[ng/µL] pipetting volume for a target mass

The first line is A = ε·c·l solved for c, with the molar extinction coefficient replaced by a mass-based factor F. Dividing by the path length converts a reading taken through any optical geometry to the 1 cm reference, and multiplying by the dilution factor D undoes whatever dilution you made before reading, so the answer describes the tube on your bench rather than the cuvette.

The unit identity in the first line is exact, not an approximation, and it is worth knowing because it removes a whole class of conversion errors: 1 µg/mL = 10⁻⁶ g ÷ 10⁻³ L = 10⁻³ g/L, and 1 ng/µL = 10⁻⁹ g ÷ 10⁻⁶ L = 10⁻³ g/L. They are the same quantity written two ways. A NanoDrop reporting 206 ng/µL and a cuvette calculation giving 206 µg/mL agree exactly; neither needs converting.

Rounding stage: FINAL ONLY. Every intermediate — the division by path length, the two multiplications, both ratios, the yield and the pipetting volume — is carried at full precision in Decimal arithmetic, and rounding to ten decimal places happens once, at the moment the result is returned. Nothing is rounded and then reused. The scientific precision of the answer is set by the photometer, not by the arithmetic: a benchtop instrument resolves about three significant figures, so 206 ng/µL is the honest way to report the worked example below, and quoting 206.0000000000 would be a lie about the measurement.

Invalid-domain behaviour is deliberate and worth stating. A negative absorbance is rejected outright, because it is a blanking artefact rather than a datum — the fix is to re-blank against your elution buffer, not to carry a negative concentration forward. A path length or dilution factor of zero is rejected because both are divisions by zero with no physical reading. An A280 or A230 of zero is NOT rejected: 'I only measured A260' is a legitimate workflow, so the corresponding ratio returns zero and the text output states plainly that it was not evaluated. Likewise an A260 of zero gives a concentration of zero, and the pipetting volume that would deliver a target mass from a zero-concentration sample is unbounded, so it returns zero with an explicit note rather than an infinity.

One approximation runs underneath all of it. The factors 50, 33 and 40 are averages over random sequence, valid for long polymers. They are not valid for short oligonucleotides, homopolymer runs, or sequences with strongly skewed base composition, where the absorbance depends on nearest-neighbour stacking and a sequence-specific coefficient is required. That is what the Custom field is for.

A worked example.

Frequently asked questions.

References& sources.

1. [1]Desjardins, P. & Conklin, D. (2010). 'NanoDrop Microvolume Quantitation of Nucleic Acids.' Journal of Visualized Experiments 45:e2565, doi:10.3791/2565. PRIMARY SOURCE, peer-reviewed, open access. Table 1 gives the conversion constants used here — DNA-50 = 50, DNA-33 = 33, RNA-40 = 40 (µg/mL per absorbance unit) — and states that pure nucleic acids typically yield a 260/280 ratio of ~1.8 for DNA and ~2.0 for RNA, with acidic solutions under-representing the ratio by 0.2–0.3 and basic solutions over-representing it by 0.2–0.3. Not independent of the NanoDrop instrument lineage; verified against a competing manufacturer below. Retrieved 2026-07-29.
2. [2]Wilfinger, W. W., Mackey, K. & Chomczynski, P. (1997). 'Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.' BioTechniques 22(3):474–481, doi:10.2144/97223st01, PMID 9067025. INDEPENDENT, peer-reviewed. Measured RNA A260/A280 rising from approximately 1.5 to 2.0 on moving the diluent from pH ~5.4 to pH 7.5–8.5, and recommends measuring in 1–3 mM Na2HPO4 at pH 8.0–8.5 for reproducibility. Abstract verified 2026-07-29; full text is paywalled at Taylor & Francis.
3. [3]DeNovix Technical Note TN 130, 'Purity Ratios Explained', revision date 2026-07-20. INDEPENDENT SECOND AUTHORITY — an instrument manufacturer separate from the NanoDrop lineage. Consulted specifically to check the primary source, and it agrees on every figure: 260/280 of ~1.8 for DNA and ~2.0 for RNA; 'generally acceptable 260/230 ratios are in the range of 2.0 – 2.2'; and the same ±0.2–0.3 pH artefact, which it independently attributes to Wilfinger et al. 1997. Retrieved and verified 2026-07-29.
4. [4]Thermo Scientific NanoDrop Technical Bulletin T042, '260/280 and 260/230 Ratios' — manufacturer technical bulletin, public mirror hosted by the University of Vermont Genetics Network. Source of the 2.0–2.2 expectation for 260/230 as a secondary purity measure. PDF retrieved 2026-07-29; its text layer could not be machine-extracted, so the figures it carries are the ones independently verified in DeNovix TN 130 above. Corroborating reference, not the load-bearing one.
5. [5]National Institute of Standards and Technology, Standard Reference Material 2372a, 'Human DNA Quantitation Standard' — certificate of analysis. STANDARDS BODY. Cited for scope rather than for a constant: NIST assigns certified values of DNA mass concentration and copy number to this material using PCR-based methods, not UV absorbance, which is the clearest available statement that A260 is an estimate of total absorbing material rather than a traceable measurement of a specific analyte. Retrieved 2026-07-29.
6. [6]International Union of Pure and Applied Chemistry, Compendium of Chemical Terminology (the 'Gold Book'), entries for 'absorbance' and the Beer–Lambert law. DEFINITIONAL AUTHORITY for A = ε·c·l and for absorbance being a dimensionless quantity referred to a stated path length. Automated retrieval of the Gold Book was blocked on 2026-07-29; the relation is standard and is restated identically in both manufacturer documents cited above.