Quantifying Oligonucleotides

Due to the very small amounts of oligonucleotide that is often produced through phosphoramidite synthesis, it is not feasible to measure the quantity of an oligonucleotide empirically. Instead, quantitation is performed by measuring the absorbance of light at 260 nm of a solution of oligonucleotide in TE buffer at pH 8.0.

One OD₂₆₀ unit is defined as the amount of oligonucleotide which gives in an absorbance reading of 1.0 when:

• The oligonucleotide is dissolved in a volume of 1.0 ml
• The oligonucleotide is measured at 260 nm in a 1 cm path-length cuvette

The OD₂₆₀ value can then be converted to micrograms or nanomoles with simple formulas if the molecular weight and extinction coefficient of the oligonucleotide are known.

Measurement of the OD₂₆₀ value is performed with a UV-Vis spectrophotometer.

• The spectrophotometer must be calibrated against know reference standards prior to use.
• The linear range of measurement must be determined for the instrument and the OD₂₆₀ values obtained must fall within that range.
• It is advisable to take repeat measurements of the oligonucleotide solution and average the data.

Converting OD₂₆₀ to Nanomoles:

• The extinction coefficient (E₂₆₀) of an oligonucleotide can be used as a factor to convert OD₂₆₀ values to nanomoles.
• The E₂₆₀ can be calculated for any sequence. However, often times a value of 33 µg / OD₂₆₀ is used for rough calculations.
• Nanomoles = ( OD₂₆₀ ÷ E₂₆₀ ) x 10⁶

Converting Nanomoles to Micrograms:

• The molecular weight of an oligonucleotide can be used as a factor to convert nanomoles to micrograms.
• Micrograms = Nanomoles x Molecular Weight x 10^-3