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DNA and RNA concentration calculator (A260)

Calculate dsDNA, ssDNA or RNA concentration from A260 absorbance, and check purity with the A260/A280 ratio.

Sample

Readings between about 0.1 and 1.0 are the most accurate.

1 for undiluted. 50 if you diluted 2 µL into 100 µL.

1 cm for a cuvette. Microvolume instruments usually report values already normalized to 1 cm.

Concentration (ng/µL, same as µg/mL)
A260/A280 purity ratio

Formula

C=A260×f×DC = A_{260} \times f \times D
How it's derived

The formula

C=A260l×f×DC = \frac{A_{260}}{l} \times f \times D

CC is the concentration in µg/mL (the same as ng/µL), A260A_{260} the absorbance at 260 nm, ll the path length in cm, DD the dilution factor, and ff a conversion factor: 50 for dsDNA, 33 for ssDNA and 40 for RNA.

Where it comes from

This is the Beer–Lambert law, A=ε l cA = \varepsilon\,l\,c, used with a shortcut. The bases in DNA and RNA have aromatic rings that absorb ultraviolet light strongly, with a peak near 260 nm.

Instead of a molar absorptivity ε\varepsilon, which would depend on the exact sequence, labs use an average mass absorptivity. Measured over many samples, a 50 µg/mL solution of double-stranded DNA has an absorbance of about 1.0 in a 1 cm cuvette. So each unit of A260A_{260} is worth 50 µg/mL. Single-stranded DNA and RNA absorb more per gram, because their bases aren’t stacked against a partner strand (“hyperchromicity”), so their factors are smaller.

The purity ratio. Proteins absorb most at 280 nm, mainly through tryptophan and tyrosine. Pure DNA has an A260/A280 of about 1.8 and pure RNA about 2.0. A lower ratio suggests protein or phenol left over from extraction.

Worked example

A dsDNA sample reads A260 = 0.45 and A280 = 0.243 in a 1 cm cuvette, undiluted:

C=0.45×50=22.5 ng/µLC = 0.45 \times 50 = 22.5\ \text{ng/µL} 0.450.243=1.85\frac{0.45}{0.243} = 1.85

That’s a clean sample.

Watch out for

  • Stay in the linear range. Readings below about 0.1 are noisy, and above about 1.0 to 1.5 they stop being proportional. Dilute and multiply back.
  • The ratio is a rough check. Glasel showed that A260/A280 is insensitive to small amounts of protein. A good ratio doesn’t prove purity.
  • RNA and free nucleotides also absorb at 260 nm and inflate DNA readings. Use a dye-based assay when accuracy matters.

Sources

  • Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual, 3rd edition (2001), Appendix 8 "Commonly Used Techniques in Molecular Cloning"
  • Glasel, J. A. (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques 18(1), 62–63.

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