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