How to use
- Enter the A260 absorbance.
- Choose the nucleic acid type. Each has its own conversion factor (dsDNA 50, ssDNA 33, RNA 40, ssOligo 33).
- If the sample was diluted for the reading, enter the dilution factor. Undiluted is 1.
- Enter the sample volume and the total amount (μg) is calculated as well. Optional.
- Enter A280 and A230 and the purity ratios A260/A280 and A260/A230 appear alongside. Optional.
Exponents can be entered with e — for example, 1.5×10⁻⁵ is entered as 1.5e-5.
Formula & notes
Nucleic acids absorb most strongly at around 260 nm, because of the ring structure of the bases. Absorbance is proportional to concentration, so multiplying by a known conversion factor gives the concentration.
- Concentration
- concentration (μg/mL) = A260 × factor × dilution
- Total amount
- amount (μg) = concentration (μg/mL) × volume (mL)
The conversion factor is the concentration at which A260 reads 1.
| Nucleic acid | Factor (μg/mL) |
|---|---|
| dsDNA (double-stranded DNA) | 50 |
| ssDNA (single-stranded DNA) | 33 |
| RNA | 40 |
| ssOligo (single-stranded oligonucleotide) | 33 |
The factors differ because stacking changes how strongly the bases absorb. In a double strand the bases are stacked against one another, so the same amount of material absorbs less.
μg/mL and ng/μL are numerically identical. 1 μg/mL = 1 ng/μL, so the figure does not change between them. Both are on screen so that the value can be copied across in whichever unit your records use.
Purity ratios
| Ratio | Reference range | Consider if low |
|---|---|---|
| A260/A280 | DNA around 1.8, RNA around 2.0 | Protein, residual phenol |
| A260/A230 | Around 2.0 – 2.2 | Salts (guanidine, acetate), residual organic solvent |
These are reference indicators, not pass or fail criteria. Whether the material works downstream depends on the sample and what it is for. A ratio outside the range does not make it unusable, and one inside does not make it clean.
Practical notes
- An A260 outside 0.1 – 1.0 is hard to trust, because it falls outside the linear range of the spectrophotometer. Dilute and read again if it is too high; concentrate, or consider a more sensitive method, if it is too low.
- Blank against the same solution the sample is dissolved in — water for water, the same TE for TE. A mismatched blank throws A230 out especially badly.
- Absorbance does not measure intact nucleic acid alone. Free nucleotides, single-stranded fragments and leftover primers all absorb at 260 nm. Where accurate quantification matters, a fluorescent dye method is better.
- Absorbance cannot tell DNA from RNA in a mixed sample. Choosing the type selects the factor for the calculation; it does not confirm what the sample contains.
- A turbid sample scatters light and reads high. Checking that A320 is close to zero is the usual precaution.