DNA Concentration Calculator

From an A260 reading to your original stock

For double-stranded DNA, a blank-corrected A260 of 1 at a 1 cm optical path corresponds to approximately 50 ng/µL. For single-stranded DNA, the conventional factor is 33. The calculator uses these factors from the NanoDrop user manual; they are estimates for the selected DNA type, rather than sequence-specific extinction coefficients.

With a reading already normalized to 1 cm, the DNA concentration formula is:

Here $F$ is 50 for dsDNA or 33 for ssDNA, and $D$ is the measurement dilution factor. The result is in ng/µL, numerically equal to µg/mL. Divide it by 1,000 to express it in mg/mL.

Suppose 2 µL of dsDNA stock was mixed with 18 µL of buffer, and the measured A260 was 0.200. The dilution factor is $(2+18)/2=10$. The measured solution contains $0.200\times50=10$ ng/µL, so the original stock contains 100 ng/µL. If 50 µL of that stock remains, its total DNA yield is $100\times50=5{,}000$ ng, or 5 µg. The volume entered in the calculator should be the available stock volume. QIAGEN's measurement guide describes the concentration-times-volume approach to yield.

Avoid correcting a NanoDrop reading twice

NanoDrop's nucleic acid A260 is reported as a 10 mm, or 1 cm, equivalent. Select Normalized to 1 cm for that reported value. Dividing it again by the instrument's short physical pedestal path would overestimate concentration.

Use Raw, unnormalized reading only when your absorbance has not already been normalized. In that case, the calculator first divides A260 by the optical path length in centimetres:

A raw A260 of 0.020 at a 1 mm path ($0.1$ cm) therefore gives the same result as a 1 cm-normalized A260 of 0.200. Enter absorbances already corrected against the sample buffer and, where appropriate, for background. This tool applies no extra A320 correction. Use a measurement dilution factor of 1 for an undiluted sample, or when the instrument has already applied that dilution correction.

Why the 260/280 ratio cannot give concentration

A260/A280 and A260/A230 compare absorbances; neither supplies the absolute A260 reading needed for concentration. For example, both 0.18/0.10 and 1.8/1.0 give a 260/280 ratio of 1.8, although their absorbances differ tenfold.

The optional purity controls divide A260 by each entered denominator. Use readings from the same sample, dilution and optical path. Values around 1.8 for DNA at 260/280 are commonly used as a reference, but buffer and pH influence them. The calculator flags ratios outside broad reference bands of 1.8–2.0 for 260/280 and 1.8–2.2 for 260/230. These are review prompts, not a sample acceptance test. The NanoDrop One guide discusses purity ratios and the importance of downstream performance.

UV absorbance does not distinguish DNA from RNA or other absorbing material. A plausible ratio can coexist with an inflated DNA estimate. Check instrument range, blanking and sample quality before treating the result as a DNA-specific measurement.

Convert DNA concentration to molarity and copy number

Mass concentration alone does not tell you how many molecules are present: a longer DNA fragment weighs more per molecule. This tool approximates molecular weight as $660\times L$ g/mol for dsDNA of length $L$ bp, or $330\times L$ for ssDNA in nucleotides. These average factors are described in Promega's Subcloning Notebook.

For concentration $C$ in ng/µL and molecular weight $MW$ in g/mol:

The conversion follows the mass-to-moles approach explained by IDT, using the exact SI Avogadro constant.

At 100 ng/µL, a 1,000 bp dsDNA fragment has a molecular weight of approximately 660,000 g/mol. That gives 151.5152 nM and about 9.124 × 10¹⁰ molecules/µL. A 5,000 bp plasmid at the same mass concentration has one fifth as many molecules: 30.3030 nM and about 1.825 × 10¹⁰ molecules/µL. Enter the full plasmid length, including its backbone.

These are intact-molecule estimates for a sample of one known length. They do not establish gene copies, cell counts, amplifiable template counts or plasmids per cell. For short primers or modified oligos, use the supplier's sequence-specific molecular weight and extinction coefficient. RNA concentration and gel/ladder densitometry require different models and are outside this calculator's scope.

Plan the next dilution

The measurement dilution above corrects a past measurement. Plan a dilution instead calculates how much original stock to use for a new preparation:

To make 100 µL at 10 ng/µL from a 100 ng/µL stock, use 10 µL stock plus 90 µL diluent. The stock aliquot contains 1,000 ng, which becomes 10 ng/µL across the final 100 µL. A dilution cannot produce a concentration higher than the stock, and the requested aliquot cannot exceed your available sample volume. Very small transfers need a suitable pipette or an intermediate dilution; the displayed arithmetic is not a validated pipetting setting.