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DNA Concentration Calculator (ng/µL from A260)

Calculate DNA concentration from spectrophotometer A260 absorbance, plus purity ratios to check for protein contamination.

Reviewed by Daniel Agrici, Founder & Lead Developer

Reviewed by Daniel Agrici, Founder & Lead Developer

Formula

Concentration (ng/uL) = A260 x Extinction Coefficient x Dilution Factor / Path Length

Where A260 is the absorbance at 260 nm, the extinction coefficient is 50 for dsDNA, 33 for ssDNA/oligos, or 40 for RNA (in ng-cm/uL), the dilution factor accounts for any sample dilution, and path length is in centimeters (typically 1 cm for standard cuvettes or 0.1 cm for microvolume instruments).

Worked Examples

Example 1: Genomic DNA Extraction Quality Check

Problem:A genomic DNA sample shows A260 = 0.45, A280 = 0.24, A230 = 0.18, measured undiluted in a 1 cm path length cuvette. Assess concentration and purity.

Solution:Concentration = A260 x 50 ng/uL x DF / path length\n= 0.45 x 50 x 1 / 1 = 22.5 ng/uL\n\nA260/A280 = 0.45 / 0.24 = 1.88 (Pure DNA: 1.7-2.0)\nA260/A230 = 0.45 / 0.18 = 2.50 (Acceptable: >2.0)\n\nTotal yield (50 uL elution) = 22.5 x 50 / 1000 = 1.125 ug

Result:Concentration: 22.5 ng/uL | A260/280: 1.88 (Pure) | A260/230: 2.50 (Clean)

Example 2: RNA Extraction from Cell Culture

Problem:RNA extracted from HeLa cells shows A260 = 1.2, A280 = 0.58, A230 = 0.55, diluted 1:10, path length 1 cm. Calculate concentration and assess quality.

Solution:Concentration = A260 x 40 ng/uL x DF / path length\n= 1.2 x 40 x 10 / 1 = 480 ng/uL\n\nA260/A280 = 1.2 / 0.58 = 2.07 (Pure RNA: 1.8-2.2)\nA260/A230 = 1.2 / 0.55 = 2.18 (Clean: 2.0-2.2)\n\nTotal yield (50 uL) = 480 x 50 / 1000 = 24 ug

Result:Concentration: 480 ng/uL | A260/280: 2.07 (Pure RNA) | A260/230: 2.18 (Clean)

Frequently Asked Questions

How does UV spectrophotometry measure DNA concentration?

UV spectrophotometry measures DNA concentration using the Beer-Lambert Law, which states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the light through the solution. Nucleic acids absorb UV light most strongly at 260 nanometers due to the aromatic ring structures of the purine and pyrimidine bases (adenine, guanine, cytosine, and thymine for DNA or uracil for RNA). An absorbance reading of 1.0 at 260 nm in a 1 cm path length cell corresponds to approximately 50 micrograms per milliliter for double-stranded DNA, 33 micrograms per milliliter for single-stranded DNA and oligonucleotides, and 40 micrograms per milliliter for RNA.

What are the limitations of spectrophotometric DNA quantification?

Spectrophotometric quantification has several important limitations that researchers should consider. It cannot distinguish between DNA, RNA, and free nucleotides, as all absorb at 260 nm. It requires relatively high concentrations, typically at least 2 to 5 nanograms per microliter, below which readings become unreliable. Contaminants that absorb near 260 nm, such as residual phenol peaking at 270 nm, can cause overestimation of DNA concentration. Turbid samples from precipitates or particulates scatter light and inflate readings. For these reasons, fluorometric methods using dyes like PicoGreen or Qubit assays are preferred when accurate quantification of low-concentration samples or specific nucleic acid types is needed for sensitive applications.

References

Reviewed by Daniel Agrici, Founder & Lead Developer · Editorial policy