DNA and RNA concentration, copy number and qPCR standards

$$c(\mathrm{ng}/\mathrm{\mu }\mathrm{L})=\frac{A_{260}\times \text{factor}\times \text{dilution}}{\text{path (cm)}}$$

The factors are the concentration that gives an absorbance of 1 at 260 nm through 1 cm: 50 ng/µL for pure double-stranded DNA (Kline & Duewer 2020, who give 37–40 for single-stranded DNA), 38 ng/µL for single-stranded DNA oligos of non-repetitive sequence and 40–80 % GC (Cavaluzzi & Borer 2004, who found that predictions from nearest-neighbour or base-composition extinction coefficients give concentrations 10–30 % too low for typical PCR primers), and 40 ng/µL for single-stranded RNA, a widely used estimate with a ±10–20 % error (Mol Ther Nucleic Acids 2024). For a protein, $c=A_{280}\times \text{dilution}/(\epsilon \times \text{path})$, using the protein’s own molar extinction coefficient.

Purity: an A260/A280 of about 1.8 is generally accepted as pure nucleic acid and 1.6 or below suggests protein, phenol or other contaminants absorbing near 280 nm; A260/A230 is commonly 2.0–2.2 for pure nucleic acid (Diagnostics 2024). Pure RNA gives ratios close to or above 2 (Sci Rep 2020).

$$\text{copies}/\mathrm{\mu }\mathrm{L}=\frac{c(\mathrm{g}/\mathrm{\mu }\mathrm{L})}{M(\mathrm{g}/\mathrm{mol})}\times N_{\mathrm{A}}$$

$M$ is the molar mass of the whole molecule, added up from the elemental formulas of the nucleotides (dAMP C₁₀H₁₄N₅O₆P, dCMP C₉H₁₄N₃O₇P, dGMP C₁₀H₁₄N₅O₇P, dTMP C₁₀H₁₅N₂O₈P and the RNA monophosphates) with the CIAAW 2021 atomic weights: each residue is a monophosphate less one water, a linear strand with 5′-OH and 3′-OH ends has one phosphate fewer than residues, and a circular strand has one per residue. A double-stranded molecule adds the complementary strand. With no sequence, equal amounts of each base are assumed. The sodium salt replaces the acidic hydrogen of each phosphate with sodium. $N_{\mathrm{A}}=6.02214076\times {10}^{23}{\mathrm{mol}}^{-1}$, exact since the 2019 redefinition of the SI (Natl Sci Rev 2020).

A plasmid miniprep reads A260 0.75, A280 0.40, A230 0.36 in a 1 cm cuvette. Concentration $=0.75\times 50=\mathbf{37.5}\mathbf{ng}\mathbf{/}\mathrm{\mu }\mathbf{L}$; A260/A280 = 1.88 and A260/A230 = 2.08, both within the ranges given for pure nucleic acid.

Copies of a 3,000 bp plasmid. As the sodium salt with equal base composition, the molar mass is $1.98559\times {10}^6\mathrm{g}/\mathrm{mol}$ (661.86 g/mol per base pair).

$$\begin{array}{rl}37.5\mathrm{ng}/\mathrm{\mu }\mathrm{L}& =\frac{37.5\times {10}^{-9}\mathrm{g}/\mathrm{\mu }\mathrm{L}}{1.98559\times {10}^6\mathrm{g}/\mathrm{mol}}\\ & =18.89\mathrm{fmol}/\mathrm{\mu }\mathrm{L}(18.89\mathrm{nM})\\ & \times 6.02214076\times {10}^{23}\\ & =\mathbf{1.137}\times {\mathbf{10}}^{\mathbf{10}}\mathbf{copies}\mathbf{/}\mathrm{\mu }\mathbf{L}\end{array}$$

Standard series. For seven 10-fold standards from ${10}^7$ copies/µL in 100 µL tubes, the first tube needs only 0.088 µL of this stock, so make an intermediate dilution first; each later tube is 10 µL of the one before plus 90 µL diluent, down to 10 copies/µL (20 copies in a 2 µL template).