OD260 concentration calculator
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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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
- Is µg/ml the same as ng/µl?
- 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.
- My A260 is above 1.0 — can I trust the 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.
- Why does my NanoDrop disagree slightly with this calculation?
- 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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