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Oligo Concentration Calculator (A260)

Calculate an oligonucleotide's concentration from its A260 and extinction coefficient or a mass factor, or convert between µM and ng/µL using the molecular weight.

Formula

c=A260×DFε ℓc = \dfrac{A_{260}\times\mathrm{DF}}{\varepsilon\,\ell}
cmass=A260×DF×Fℓc_{\mathrm{mass}} = \dfrac{A_{260}\times\mathrm{DF}\times F}{\ell}
cmass=cmol×MWc_{\mathrm{mass}} = c_{\mathrm{mol}} \times \mathrm{MW}
ε\varepsilon
the oligo's molar extinction coefficient at 260 nm, in M⁻¹ cm⁻¹, from its data sheet
FF
33 µg/mL per A260 for single-stranded DNA, 40 for RNA
MW\mathrm{MW}
molecular weight, in g/mol

How it works

By the Beer-Lambert law, absorbance is the extinction coefficient times the concentration times the path length. Dividing the measured absorbance, corrected for any dilution, by the extinction coefficient gives the molar concentration directly.

An oligo's extinction coefficient depends on its sequence, because the bases absorb differently and stacking lowers the absorbance. Vendors calculate it from the sequence and print it on the data sheet; use that value when you have it. When you do not, the average mass factor of about 33 µg/mL per absorbance unit for single-stranded DNA, together with the molecular weight, gives an estimate.

Worked example

A 1:100 dilution of an oligo stock reads A260 = 0.25 in a 1 cm cuvette; the data sheet gives ε = 200,000 M⁻¹ cm⁻¹.

  1. A260 of the stock = 0.25 × 100 = 25.
  2. c = 25 / (200,000 M⁻¹ cm⁻¹ × 1 cm) = 1.25 × 10⁻⁴ M.

The stock is 125 µM.

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

Assumptions

  • The reading is blank-corrected and in the linear range of the instrument, roughly A260 of 0.1 to 1.
  • The sample is a single oligo with no other nucleic acid or absorbing contaminant.
  • The extinction coefficient is for the sequence, in the buffer and at the temperature used. Base-pairing and secondary structure lower the absorbance.

Common mistakes

  • Forgetting the dilution factor, which gives the diluted sample's concentration rather than the stock's.
  • Using the ε of a different oligo, or the average mass factor, for a short or unusual sequence and trusting the answer to more digits than it deserves.
  • Measuring an oligo that is partly double-stranded or folded, which reads lower than the single-stranded value.

Related tools

Oligo Resuspension and Dilution CalculatorBuffer volume for a stock, and dilution to a working concentrationOpen calculatorDNA and RNA Concentration Calculator (A260)Nucleic acid concentration and purity from absorbanceOpen calculatorDNA and RNA Molecular Weight CalculatorExact molecular weight of an oligo or sequence, ss or dsOpen calculatorConcentration Unit ConverterMolar, mass per volume, mg/dL and % w/vOpen calculator

Related equipment

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