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DNA/RNA concentration and purity (A260)

Absorbance at 260 nm to a concentration (ng/µL), and purity read from the 260/280 and 260/230 ratios, with their thresholds.

Parameters

Sample
1 for a measurement on the neat sample; 10 if you diluted tenfold.
Absorbances read
Optional; without it, the ratio is not computed.

Spectrum

three measurements
double-stranded DNA · dilution 1C = 50 ng/µL23026028000.51nmA0.47610.556260/280 ratio1.621.8expected260/230 ratio1.82.42.1expected
Calculation
C = A260 × 50 × 1 = 1 × 50 × 1 = 50 ng/µL
Ratios
A260/A280 = 1 / 0.556 = 1.7986
A260/A230 = 1 / 0.476 = 2.1008
Notes (2)

The calculation uses only the three wavelengths entered, with no interpolation.

The grey band is the reference range for the classification; outside it does not mean unusable.

Result

continuous calculation
Signature quantity
C50ng/µLboth ratios within their band
260/280 ratio
1.7986 (expected)
260/230 ratio
2.1008 (expected)
Conversion factor
50 µg/mL
Dilution factor
1

The undiluted sample contains 50 ng/µL.

Calculation, assumptions and sources

OverviewOne absorbance unit at 260 nm corresponds to 50 µg/mL of double-stranded DNA, 40 for RNA and 33 for single-stranded DNA; the concentration accounts for the dilution factor.

One absorbance unit at 260 nm corresponds to 50 µg/mL of double-stranded DNA, 40 for RNA and 33 for single-stranded DNA; the concentration accounts for the dilution factor. The 260/280 and 260/230 ratios flag contaminants, with the published thresholds (1.8 for DNA, 2.0 for RNA). A ratio without a measured absorbance at 280 or 230 nm is not assessed.

Method & formulasC (µg/mL) = A260 × factor × dilution
C (µg/mL) = A260 × factor × dilutionfactors: 50 (dsDNA), 40 (RNA), 33 (ssDNA)purity: 260/280 ≈ 1.8 (DNA), 2.0 (RNA); 260/230 ≈ 2.0-2.2

Beer-Lambert law at 260 nm, in a 1 cm cuvette: conventional factors convert absorbance into mass concentration. ng/µL and µg/mL are the same unit.

A260
the absorbance at 260 nm, the absorption maximum of nucleic bases.
260/280 ratio
sensitive to proteins and phenol, which absorb at 280 nm. Expected around 1.8 for DNA, 2.0 for RNA.
260/230 ratio
sensitive to chaotropic salts, EDTA and phenol, which absorb at 230 nm. Expected between 2.0 and 2.2.
Dilution factor
the ratio between the sample and the solution measured. Diluted tenfold, the true concentration is ten times the reading.
Validity domainThe factors assume an average base composition and a 1 cm cuvette; for a short oligonucleotide, the extinction coefficient of the sequence is more accurate.

The factors assume an average base composition and a 1 cm cuvette; for a short oligonucleotide, the extinction coefficient of the sequence is more accurate. Photometry is reliable between about 0.1 and 1 absorbance unit. Above, dilute; below, noise dominates and a fluorometric intercalating-dye assay is better suited.

Purity ratios and contaminantsA low 260/280 flags residual proteins from a phenol-chloroform extraction; a low 260/230, the guanidine salts of column kits or the EDTA of the buffer.

A low 260/280 flags residual proteins from a phenol-chloroform extraction; a low 260/230, the guanidine salts of column kits or the EDTA of the buffer. Remedies: rewash the column, reprecipitate, or change the blank. Good ratios do not guarantee integrity; free nucleotides and degraded DNA also absorb at 260 nm, and only electrophoresis checks it.