BioDeviceHub

DNA and RNA Concentration Calculator (A260)

Convert an A260 absorbance reading into a DNA or RNA concentration in ng/µL, and check purity with the A260/A280 and A260/A230 ratios.

Formula

C=A260×F×DFℓC = \dfrac{A_{260}\times F\times \mathrm{DF}}{\ell}
purity ratios:A260A280,  A260A230\text{purity ratios:}\quad \dfrac{A_{260}}{A_{280}},\ \ \dfrac{A_{260}}{A_{230}}
CC
concentration, in µg/mL (equal to ng/µL)
FF
50 for double-stranded DNA, 33 for single-stranded DNA, 40 for RNA, in µg/mL per absorbance unit
ℓ\ell
optical path, in cm (1 for a standard cuvette)

How it works

Nucleic acids absorb ultraviolet light most strongly near 260 nm. At a 1 cm path, one absorbance unit corresponds to about 50 µg/mL of double-stranded DNA, 33 µg/mL of single-stranded DNA or 40 µg/mL of RNA, so the reading scales directly to a concentration.

Proteins absorb near 280 nm, and salts, phenol and carbohydrates near 230 nm. Clean DNA typically gives an A260/A280 of about 1.8 and clean RNA about 2.0, with A260/A230 usually between 2.0 and 2.2. Lower ratios point to contamination.

Worked example

A double-stranded DNA sample diluted 1:20 reads A260 = 0.25 and A280 = 0.135 in a 1 cm cuvette.

  1. C = 0.25 × 50 µg/mL × 20 / 1 cm = 250 µg/mL.
  2. A260/A280 = 0.25 / 0.135 = 1.85.

The stock is 250 ng/µL, with a purity ratio consistent with clean DNA.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The blank has been subtracted and the reading is in the linear range of the instrument.
  • The sample is a single species. The conversion factors are averages and differ for short oligonucleotides, whose absorbance depends on sequence.
  • Absorbance cannot distinguish DNA from RNA. Both contribute to A260.

Common mistakes

  • Forgetting the dilution factor, which gives the concentration of the diluted sample rather than the stock.
  • Measuring in water. The A260/A280 ratio depends on pH, so use a lightly buffered solution for purity checks.
  • Reading a very dilute sample. Below about 0.1 absorbance the measurement is dominated by noise.

Related tools

DNA Dilution CalculatorDilute a DNA or RNA stock to a working concentrationOpen calculatorCalibration Curve Calculator (Standard Curve)Linear fit, residuals and an unknown with its uncertaintyOpen calculatorPCR Master Mix CalculatorComponent volumes for any number of reactionsOpen calculatorGC Content CalculatorGC percentage and base composition of a sequenceOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.