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A UV spectrophotometer estimates DNA concentration by measuring how much 260 nm light a sample absorbs. For double-stranded DNA, an A260 of 1.0 corresponds to about 50 µg/mL, so a single reading, corrected for background and dilution, gives a concentration. This guide covers the formula, a worked example, how to read the purity ratios, and how cuvette and plate-reader formats differ. Used by molecular biology labs, core facilities, and students checking extractions before PCR or sequencing.
What it is
A spectrophotometer for DNA concentration is any UV-capable absorbance instrument that can read at 260 nm: a benchtop cuvette instrument, a microvolume instrument, or a microplate reader with UV capability. Nucleic acids absorb strongly at 260 nm, and the absorbance scales with the amount of DNA in the light path. For the underlying physics and blanking, see UV-Vis spectrophotometer basics; for the small-volume instrument class, see what a microvolume spectrophotometer is.
How an A260 reading becomes a concentration
Promega gives the working formula: concentration (µg/mL) = (A260 reading − A320 reading) × dilution factor × 50 µg/mL. The 320 nm reading is a background term: Promega notes that absorbance there indicates turbidity, so subtracting it removes light scattering from the A260 value. Promega and QIAGEN both state that an A260 of 1.0 equals 50 µg/mL of double-stranded DNA (1 cm path, neutral pH).
Total yield follows from the concentration: DNA yield (µg) = concentration (µg/mL) × total sample volume (mL) (Promega).
Worked example (illustrative numbers)
Suppose a sample diluted 1:10 reads A260 = 0.45 and A320 = 0.05. Concentration = (0.45 − 0.05) × 10 × 50 = 200 µg/mL (200 ng/µL). If the undiluted preparation totals 0.1 mL, the yield is 200 × 0.1 = 20 µg. The values are invented to show the arithmetic only.
Conversion factor by nucleic acid
BMG LABTECH lists the factors for an A260 of 1.0 in a cuvette: double-stranded DNA 50 µg/mL, single-stranded DNA 33 µg/mL, RNA 40 µg/mL. Using the 50 factor on a single-stranded or RNA-rich sample gives the wrong number. QIAGEN adds that the method does not differentiate between DNA and RNA.
Types: cuvette, microvolume and plate reader
| Format | Key points | Source |
|---|---|---|
| Cuvette instrument | 1 cm path; use a quartz cuvette for UV; if several cuvettes are used they must be matched; zero the instrument with the buffer used for the sample | QIAGEN Bench Guide |
| Microplate reader | Path length is shorter, so the same DNA reads lower (about 0.7 OD where a cuvette gives 1.0 OD); path length correction makes results comparable with a cuvette | BMG LABTECH |
Microvolume instruments read about a microlitre without a cuvette; the design is covered in the microvolume spectrophotometer guide, and plate readers in what a plate reader is.
Specifications that matter
- Linear range. Keep A260 between 0.1 and 1.0 (Promega, QIAGEN). Dilute strong samples into range and multiply back with the dilution factor.
- Path length. The 50 µg/mL factor assumes a 1 cm path (QIAGEN). Other paths need correction (BMG LABTECH).
- Wavelength scan. QIAGEN advises a 220–320 nm scan; a clean DNA sample shows a smooth curve with a peak at 260 nm.
Reading the purity ratios
| Reading | Guidance | Source |
|---|---|---|
| A260/A280 | 1.7–2.0 for good-quality DNA; about 1.6 acceptable for most uses; lower suggests contaminants | Promega |
| A260/A280 | Pure DNA 1.7–1.9 | QIAGEN |
| A260/A280 | Greater than about 1.7 is called pure | BMG LABTECH |
| A260/A230 | Best if greater than 1.5; a strong 230 nm signal points to organic compounds or chaotropic salts | Promega |
| A320 | A reading here indicates turbidity | Promega |
Published guidance differs on the exact upper bound (2.0 versus 1.9), so treat the ratio as a range, not a pass-fail line. Buffer matters: QIAGEN reports the A260/A280 ratio drops at lower pH, and unbuffered water gives values below 1.8 that vary. For ratio measurements, QIAGEN suggests a slightly alkaline buffer such as 10 mM Tris-Cl, pH 7.5, and a low-salt neutral buffer (for example Tris-Cl, pH 7.0) for dilution. For more on purity ratios in extraction work, see DNA extraction protocol basics.
How to choose: absorbance or fluorescence
QIAGEN states that absorbance is best for microgram quantities and that for small amounts, such as PCR products, an agarose gel may work better. BMG LABTECH notes that A260 has low sensitivity and is affected by nucleotides, single-stranded nucleic acids and preparation compounds, while fluorescent methods are much more sensitive and specific for DNA. A side-by-side view is in Qubit vs NanoDrop.
Safe use and handling basics
- Blank (zero) the instrument with the same buffer the DNA is in (QIAGEN).
- Use quartz cuvettes for UV readings and keep multiple cuvettes matched (QIAGEN).
- Dilute in a low-salt, neutral buffer rather than unbuffered water (QIAGEN).
- Follow the instrument maker’s instructions for cleaning and for any hazardous solvents carried over from extraction.
Common mistakes
- Ignoring RNA, protein or guanidine. RNA absorbs at 260 nm, protein at 280 nm, and guanidine raises the 260 reading, any of which can make the DNA quantity look higher than it is (Promega).
- Phenol carryover. Phenol absorbs near 270–275 nm and can mimic both higher yield and higher purity (QIAGEN).
- Reading outside 0.1–1.0. Readings outside the range are unreliable; dilute and re-read (Promega, QIAGEN).
- Skipping the A320 term. A turbid sample inflates A260 unless background is subtracted (Promega).
- Comparing cuvette and plate numbers directly. Without path length correction, plate readings run lower (BMG LABTECH).
FAQ
How do you calculate DNA concentration from absorbance?
Subtract A320 from A260, multiply by the dilution factor, then by 50 µg/mL for double-stranded DNA (Promega).
What does an A260/A280 ratio of 1.8 mean?
It falls inside the ranges both Promega (1.7–2.0) and QIAGEN (1.7–1.9) give for good-quality DNA.
Can a spectrophotometer tell DNA from RNA?
No. QIAGEN states the method does not differentiate between them.
Is a plate reader as accurate as a cuvette instrument?
Readings are comparable only after path length correction (BMG LABTECH).
Sources
- Promega, How do I determine the concentration, yield and purity of a DNA sample? (checked 2026-10-29)
- QIAGEN Bench Guide, Spectrophotometric measurement of DNA concentration (checked 2026-10-29)
- BMG LABTECH, UV absorbance DNA quantitation (checked 2026-10-29)
