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Oligonucleotide Quantitation Using Spectrophotometry

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05/13/2014

Oligonucleotide Quantitation Using Spectrophotometry

Spectrophotometry allows for the quantitative measurement of molecular compounds or substances by measuring the quantitative reflection or transmission properties of matter as a function of a specific wavelength. For example, oligonucleotides are quantified by measuring the absorbance of ultraviolet light (UV) of a given oligonucleotide sample using a spectrophotometer.

One optical density unit, or 1 O.D., typically equals 50 pg/ml of double stranded DNA; 33 pg/ml of single stranded DNA; or 40 pg/ml of single stranded RNA.

Usually, automated synthesis of oligonucleotides is performed at scales described in micromoles. Standard synthesis protocols can range from 200 nanomoles to 50 µmoles per oligonucleotide synthesized.

The scale of a synthesis can be converted to units of weight (microgram, milligram, or gram) by using the following formula:

The average molecular weight of a deoxynucleotide base or 1-mer, is 325,

Therefore,

1 µmol of a 25-mer oligonucleotide = 325 x 25 g / 106 = 0.081 g = 8.1mg

And,

8.1 mg = 8100 µg / 33 = 245 O.D. (for single stranded DNA)