The concentration formula, and the factor that changes with sample type
The 50 is the conversion factor for double-stranded DNA. Swap it for the value that matches your sample: single-stranded DNA absorbs less, RNA sits in between. Multiply by the dilution factor because the reading is taken on the diluted aliquot, not the stock.
| Sample type | Factor (µg/mL per A260) |
|---|---|
| Double-stranded DNA | 50 |
| Single-stranded DNA | 33 |
| RNA | 40 |
To read in ng/µL instead, the numbers are identical: A260 of 0.5 on dsDNA is 25 µg/mL, which is the same as 25 ng/µL.
The 260/280 ratio tells you whether the number is trustworthy
A concentration is only meaningful if the sample is clean. The ratio of absorbance at 260 nm to 280 nm flags protein, phenol and RNA carryover.
| A260/A280 | What it means |
|---|---|
| 1.8 | Clean DNA |
| ~2.0 | Clean RNA |
| Below 1.7 | Protein or phenol contamination |
| Above 2.0 (for DNA) | Likely RNA carryover |
What throws the reading off
- Phenol. It absorbs around 270 nm and inflates the A260. Remove all phenol before measuring.
- RNA in a DNA prep. The method cannot tell DNA from RNA, so contaminating RNA reads as extra DNA. Treat with RNase if it matters.
- Total yield. To get the amount of nucleic acid, multiply concentration by sample volume: yield (µg) = concentration (µg/mL) × volume (mL).
Common questions
What does A260 = 1.0 mean?
An absorbance of 1.0 at 260 nm corresponds to 50 ug/mL of pure double-stranded DNA in an undiluted sample. That is the standard spectrophotometric conversion.
Why is the factor 50 for DNA but 40 for RNA?
The factor reflects how strongly each nucleic acid absorbs at 260 nm. Double-stranded DNA uses 50, single-stranded DNA uses 33, and RNA uses 40 ug/mL per absorbance unit. Pick the factor that matches your sample.
My A260/A280 ratio is 1.5. Is that a problem?
Yes. A ratio below 1.7 usually means protein or phenol contamination. Pure DNA reads 1.8 and pure RNA reads around 2.0. Re-purify the sample before trusting the concentration.
Do I need to keep the reading below 1.0?
The most accurate range is A260 between 0.1 and 1.0. If the raw reading is above 1.0, dilute the sample and multiply the result by the dilution factor.
