What this calculator does
Nucleic acid concentration is read from absorbance at 260 nm. One absorbance unit corresponds to about 50 µg/mL of double-stranded DNA, 33 of single-stranded DNA and 40 of RNA, and those three conversion factors are the whole of the calculation once the dilution factor is applied.
The factor is the thing to get right. Using the dsDNA figure of 50 on an RNA sample overstates the concentration by about 25%, and on single-stranded DNA by about 50%. The difference arises because single strands absorb more per unit mass than the stacked bases of a duplex, an effect known as hypochromicity.
The formula
The A260 reading is multiplied by the conversion factor for the nucleic acid type and by the dilution factor, giving concentration in µg/mL, which is numerically the same as ng/µL. Total yield multiplies that concentration by the sample volume. The calculation assumes a 1 cm path length, which is standard for cuvettes and is corrected for automatically on microvolume instruments.
| Term | Meaning |
|---|---|
| A260 | Absorbance at 260 nm, where nucleic acids absorb most strongly. |
| Conversion factor | 50 µg/mL per A260 for dsDNA, 33 for ssDNA, 40 for RNA. |
| Dilution factor | How much the sample was diluted before reading, which multiplies back up to the original concentration. |
| A260/A280 | A separate purity ratio, around 1.8 for clean DNA and 2.0 for clean RNA. Not calculated here. |
The inputs explained
| Field | What to enter |
|---|---|
| A₂₆₀ reading | The A260 absorbance reading. Readings above about 1.0 are outside the reliable linear range on most instruments and the sample should be diluted further. |
| Dilution factor | The dilution factor. Enter 1 if the sample was read undiluted. |
| Sample type | Nucleic acid type, which sets the conversion factor. |
| Sample volume (µL) | Sample volume in microlitres, used only for the total yield figure. |
When to use it
Quantifying a prep before downstream work
Almost every subsequent step, from restriction digests to library preparation, specifies an input mass, so the concentration has to be known first.
Checking a yield against expectations
Total yield against the starting material is the quickest indication of whether an extraction worked as it should have.
Normalising samples to a common concentration
Comparing samples requires equal input, and the concentration figure is what the dilution is calculated from.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
How does nucleic acid type change the concentration?
The same absorbance reading interpreted as each of the three nucleic acid types.
| Nucleic acid type | Concentration (ng/µL) | Total yield | Conversion factor used |
|---|---|---|---|
| Double-stranded DNA | 42.50 ng/µL | 2.1 µg | 50 µg/mL per A₂₆₀ |
| Single-stranded DNA | 28.05 ng/µL | 1.4 µg | 33 µg/mL per A₂₆₀ |
| RNA | 34.00 ng/µL | 1.7 µg | 40 µg/mL per A₂₆₀ |
Questions
Why is the dsDNA factor 50 and the RNA factor 40?
Because bases stacked in a double helix absorb less per unit mass than free ones, an effect called hypochromicity. Single-stranded nucleic acids absorb more strongly, so a lower mass gives the same absorbance, which is why their conversion factors are lower.
What does the A260/A280 ratio tell me?
Purity rather than quantity. Around 1.8 indicates reasonably clean DNA and around 2.0 clean RNA. Substantially lower suggests protein or phenol contamination. It is a separate reading from the one this calculator uses and does not affect the concentration arithmetic.
My reading is above 1.0. Is that a problem?
Usually yes. Most spectrophotometers are only reliably linear up to around 1.0 absorbance units, and readings above that underestimate concentration. Dilute the sample further and enter the dilution factor rather than trusting a high reading.
Is ng/µL the same as µg/mL?
Numerically identical, since both are one part in a million by the same ratio. The calculator reports both because instruments and protocols use the two interchangeably and seeing them side by side avoids a units mistake.
To convert that concentration into a copy number, see the DNA copy number calculator. For setting up a ligation from measured concentrations, see the ligation ratio calculator.