Why is a different factor used to calculate the concentration of double-stranded DNA than for calculating the concentration of single-stranded DNA?
Factor F is calculated from the molar absorbance coefficient of a molecule in solution and the optical path length of the cuvette used (see Beer-Lambert law). The absorption behavior of nucleic acids is influenced by the aromatic rings of the bases. Here the molar absorbance coefficient of a double-stranded nucleic acid molecule with which the bases are in close contact with each other, is smaller than of a single-stranded nucleic acid molecule. It thus follows that single-stranded nucleic acid molecules have a higher rate of absorption than double-stranded molecules (Sambrook et al. 2001. Molecular Cloning 3rd edition, A8.20). With an optical path length of 10 mm and under neutral to slightly alkaline measuring conditions an optical density of 1 at 260 nm thus corresponds to approx. 50 µg/ml of double-stranded DNA, 37 µg/ml of single-stranded DNA, 40 µg/ml of RNA and appr. 30 µg/ml of oligonucleotides.
