OD260 concentration calculator
Maintained by the Talindrew team · Last updated
Nucleic acid concentration from absorbance at 260 nm is the A260 reading times a conversion factor times any dilution factor: 50 µg/ml per A260 unit for double-stranded DNA, 40 for RNA, 33 for single-stranded DNA (1 cm path). An A260 of 0.5 from a 10× diluted dsDNA sample is 250 µg/ml.
Result
c = A260 × factor × dilution, 1 cm path. Factors: dsDNA 50, RNA 40, ssDNA 33 µg/ml per A260 unit (Sambrook & Russell; Promega BioMath).
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What it looks like
Formula
c (µg/ml) = A260 × factor × dilution (dsDNA 50, RNA 40, ssDNA 33)
Source: Sambrook & Russell, Molecular Cloning; Promega BioMath
Worked example
Given: dsDNA sample diluted 10×, measured A260 = 0.5
- 1.Apply the dsDNA factor: 0.5 × 50 = 25 µg/ml in the cuvette.
- 2.Undo the dilution: 25 × 10 = 250 µg/ml in the stock.
Stock concentration = 250 µg/ml (250 ng/µl)
How the calculation flows
Units & constants
| dsDNA | 1 A260 = 50 µg/ml |
|---|---|
| RNA | 1 A260 = 40 µg/ml |
| ssDNA | 1 A260 = 33 µg/ml (Promega; some sources use 37–38) |
| Path length | 1 cm (spectrophotometers normalise to this) |
| Purity check | A260/A280 ≈ 1.8 for clean DNA, ≈ 2.0 for RNA |
| Reliable range | A260 between ~0.1 and 1.0: dilute to stay within it |
| Price | Free |
Where do the 50/40/33 factors come from?
They are rearranged Beer–Lambert averages: A = ε·c·l, with the average extinction coefficient per microgram of each species at 260 nm. Double-stranded DNA absorbs less per mass than single strands because base stacking in the duplex hypochromically suppresses absorbance, which is why the ssDNA factor (33) is smaller than 50: the same A260 means less mass of ssDNA.
What does A260/A280 tell you?
Protein absorbs at 280 nm, so the 260/280 ratio flags protein contamination: ~1.8 is clean DNA, ~2.0 clean RNA, and values below ~1.6 suggest protein or phenol carryover. A low 260/230 ratio points instead at guanidine, phenol or carbohydrate. Ratios diagnose purity; the A260 value itself gives concentration.
Should you use the flat factor for oligos?
For anything quantitative with short oligos, primer stocks, probes, prefer the sequence-specific extinction coefficient, because base composition swings the true factor for a short oligo well away from any average. The flat 33 µg/ml factor is fine for rough checks on longer ssDNA.
Frequently asked questions
Yes, numerically identical: 250 µg/ml = 250 ng/µl. Spectrophotometer software usually reports ng/µl; the formula gives µg/ml; they are the same number.
Not really. Most spectrophotometers are linear roughly between A260 0.1 and 1.0. Dilute the sample so the reading falls in range, then multiply by the dilution factor: the calculator's dilution field does this for you.
Short-path instruments measure at 0.05–1 mm and normalise to 1 cm internally, and some apply slightly different ssDNA factors (33 vs 37). Check which factor and normalisation your instrument uses: the conventions, not the physics, differ.
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