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Nucleic Acid Concentration Calculator

Chemistry

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Sample

Single strands absorb more per µg than duplexes (hypochromicity), which is why ssDNA reads 33 and dsDNA 50.

Sample type

Conversion factor (µg/mL per A260 unit)

Pre-filled with the standard value for this type. Some protocols use 37 for ssDNA.

Readings

Enter absorbances as read. A slightly negative value after blanking is fine.

A260 (required)

A280 (optional)

A230 (optional)

Background A320 (optional)

Background wavelength

Use whichever wavelength your instrument corrects at. It is subtracted from every other reading.

Pathlength and dilution

Pathlength

Dilution factor (DF)

1 means undiluted; 20 means 1:20.

Molecule (optional)

Needed for molar concentration, copies and ends.

Length (bp)

A whole number. 3.1e9 and digit grouping are accepted.

Topology

5′ ends

Presets

MW override (g/mol, optional)

Replaces the computed molecular weight.

Mass basis (affects MW, molar values and copies; never ng/µL)

Matches NEBioCalculator to within 0.01 %.

Optional extras

Sample volume (µL)

Gives the total yield.

Significant figures

Enter the A260 reading, or pick an example above, to see the concentration.

Related: the Standard Curve Calculator for fluorometric (Qubit, PicoGreen) quantitation, and the Avogadro's Number Calculator for general mole to count conversion.

About This Tool

Nucleic Acid Concentration Calculator – From A260 to ng/µL, Copies and Purity

Enter the absorbance readings from a spectrophotometer and this nucleic acid concentration calculator returns the concentration of a DNA, RNA or oligonucleotide sample, the A260/A280 and A260/A230 purity ratios with a verdict, and the same amount as a molar concentration, copies per µL and pmol of ends. Everything is calculated in your browser.

Why nucleic acids absorb at 260 nm

The purine and pyrimidine bases absorb ultraviolet light with a maximum near 260 nm. The Beer–Lambert law links that absorbance to concentration: A = ε · c · l. For a mixed sample you rarely know ε, so a single conversion factor is used instead.

Where 50, 33 and 40 come from

One absorbance unit at 260 nm over a 1 cm path corresponds to about 50 µg/mL of double-stranded DNA, 33 µg/mL of single-stranded DNA and 40 µg/mL of RNA. The differences come from hypochromicity: stacked base pairs absorb less than free bases, so a duplex needs more mass than a single strand to give the same reading. These factors are averages over typical base composition, which is why the factor field is editable.

Oligonucleotides: why the sequence matters

A short oligo has too few bases for an average to be reliable, so the tool computes its ε260 from the sequence with the nearest-neighbour method. Each pair of adjacent bases contributes its own absorbance, and the single bases inside the chain are subtracted once. The concentration is then A260 × DF / (ε260 × l). A dye or other modification adds absorbance that the sequence cannot predict, so use the supplier's ε260 in the override field when you have it.

Reading 260/280 and 260/230

A 260/280 ratio near 1.8 is typical of pure DNA and near 2.0 of pure RNA. Lower values suggest protein or phenol. A 260/230 ratio of 2.0 to 2.4 is expected for a clean sample; lower values point to guanidine salts, EDTA, phenol, carbohydrate or TRIzol carry-over. The ratios depend on pH, reading lower in unbuffered water, so measure in a buffer such as TE. They are also unreliable at low concentration, below roughly 10 ng/µL of dsDNA.

Pathlength, dilution and the linear range

The tool corrects for the background reading, multiplies by the dilution factor and divides by the pathlength, so a 1:20 dilution read in a 1 cm cuvette is handled exactly. Most cuvette instruments are reliable for corrected A260 between about 0.1 and 1.5. Microvolume instruments switch their own pathlength and display a value normalised to 10 mm, so the linear-range check is skipped for them.

From ng/µL to copies

Copy number follows from the molecular weight: copies/µL = ng/µL × 10⁻⁹ × N_A / MW. For a double-stranded fragment of N base pairs the free-acid weight is about 617.9 × N g/mol. Many calculators use the sodium salt instead, about 661.9 g/mol per bp, which is where the rules of thumb of 650 and 660 come from. Choosing the sodium-salt basis changes the molar value and the copy number by about 7 percent, and never changes ng/µL.

Absorbance measures total nucleic acid, not intact template.
Degraded DNA, RNA in a DNA prep and free nucleotides all absorb at 260 nm, so a good-looking number does not guarantee amplifiable or intact material.

When to use a fluorometric assay instead

A260 cannot tell DNA from RNA or from free nucleotides. For low-concentration samples, or when RNA contamination matters, a dye-based assay such as Qubit or PicoGreen is more specific; use a standard curve to convert its signal to a concentration.

Frequently Asked Questions

Is the Nucleic Acid Concentration Calculator free?

Yes, Nucleic Acid Concentration Calculator is totally free :)

Can I use the Nucleic Acid Concentration Calculator offline?

Yes, you can install the webapp as PWA.

Is it safe to use Nucleic Acid Concentration Calculator?

Yes, any data related to Nucleic Acid Concentration Calculator only stored in your browser (if storage required). You can simply clear browser cache to clear all the stored data. We do not store any data on server.

How do I calculate DNA concentration from A260?

Subtract the background reading (usually A320) from A260, multiply by the dilution factor and divide by the pathlength in cm, then multiply by the conversion factor: 50 ng/µL per absorbance unit for double-stranded DNA, 33 for single-stranded DNA and 40 for RNA. For example, a corrected A260 of 0.511 on a 1:20 dilution in a 1 cm cuvette gives 0.511 × 20 × 50 = 511 ng/µL.

Why are the conversion factors 50, 33 and 40 different for dsDNA, ssDNA and RNA?

Stacked base pairs in a duplex absorb less light than the same bases in a single strand (hypochromicity), so a given mass of double-stranded DNA gives a lower A260 than single-stranded DNA. That is why 1 A260 unit equals 50 µg/mL of dsDNA but only 33 µg/mL of ssDNA, and 40 µg/mL of RNA. They are averages, so a sequence with unusual base composition can deviate by several percent; the factor field is editable for that reason.

What do the A260/A280 and A260/A230 ratios tell me about purity?

A260/A280 of about 1.8 is typical for pure DNA and about 2.0 for pure RNA; lower values point to protein or phenol. A260/A230 should be 2.0 to 2.4; lower values suggest carry-over of guanidine salts, EDTA, phenol, carbohydrate or TRIzol. Both ratios are unreliable when the sample is dilute (below roughly 10 ng/µL), and A260/A280 reads lower in unbuffered water than in a buffer at pH 7.5 to 8.5.

How do I convert ng/µL to copies per µL?

Divide the mass concentration by the molecular weight and multiply by Avogadro's number: copies/µL = ng/µL × 10⁻⁹ × 6.022 × 10²³ / MW. The molecular weight comes from the length (about 617.9 g/mol per base pair of dsDNA as the free acid) or from the sequence for an oligo. For example, 62.5 ng/µL of a 500 bp linear fragment is about 1.2 × 10¹¹ copies/µL.

Why do copy-number calculators disagree (617.9 vs 650 vs 660 g/mol per bp)?

The figure depends on what is counted. The free acid of an average base pair is about 617.9 g/mol; the sodium salt, with one Na⁺ in place of each phosphate H⁺, is about 661.9 g/mol, which is where the rule-of-thumb 650 and 660 values come from. Switching between them changes the copy number by about 7 percent, so pick the basis that matches the calculator you are comparing with.

How is an oligo's concentration calculated from its sequence?

The tool estimates the extinction coefficient ε260 from the sequence with the nearest-neighbour method, then applies the Beer–Lambert law: concentration (M) = A260 × dilution / (ε260 × pathlength). Multiplying by the molecular weight gives ng/µL. A dye or other modification adds absorbance that the sequence alone does not capture, so enter the supplier's ε260 in the override field when you have it.