How to calculate DNA and RNA concentration from A260
Nucleic acids absorb ultraviolet light strongly at 260 nm. Measuring that absorbance with a spectrophotometer such as a NanoDrop is the quickest way to estimate how much DNA or RNA you have, and two ratios tell you about purity.
The conversion factors
At a 1 cm path length, an absorbance of 1.0 at 260 nm corresponds to approximately:
| Nucleic acid | ng/µL per A260 unit |
|---|---|
| Double-stranded DNA | 50 |
| RNA | 40 |
| Single-stranded DNA | 33 |
| Oligonucleotides | ~20–30 (sequence dependent) |
concentration (ng/µL) = A260 × conversion factor × dilution factor
Example: a dsDNA sample diluted 1:10 reads A260 = 0.42. Concentration = 0.42 × 50 × 10 = 210 ng/µL.
Purity ratios
- A260/280 around 1.8 suggests clean DNA, around 2.0 clean RNA. Lower values point to protein or phenol contamination.
- A260/230 should be about 2.0–2.2. Low values suggest guanidine salts, EDTA, carbohydrates or phenol, common carry-overs from column kits and extractions.
A ratio that is off doesn't always mean the sample is unusable, but it can inhibit enzymes like polymerases and ligases, and it makes the concentration estimate less reliable.
Limitations
- A260 can't tell DNA from RNA, or intact DNA from free nucleotides. RNA in a DNA prep inflates the result; RNase treatment helps.
- Readings below about 0.1 are unreliable. For dilute samples, or when you need an accurate number (like NGS libraries), use a fluorometric assay such as Qubit, which binds specifically to dsDNA or RNA.
- Blank with the same buffer your sample is in. Elution buffers containing EDTA or Tris absorb slightly and shift the result.
Frequently asked questions
Why is my NanoDrop reading higher than Qubit?
Absorbance also measures RNA, free nucleotides, primers and single-stranded DNA, while dsDNA fluorescent dyes measure only double-stranded DNA. Differences of 1.5–3 times are common in crude preps.
What does an A260/280 ratio above 2.0 mean for DNA?
Often RNA contamination, since RNA has a higher ratio than DNA. It can also come from a wrong blank or a very dilute sample.
Do I need to correct for path length on a NanoDrop?
No. The instrument normalises its readings to a 1 cm path length automatically.