DNA and RNA concentration, copy number and qPCR standards
Enter an A260 and the calculator gives the concentration (dsDNA 50, RNA 40 or ssDNA 38 ng/µL per absorbance unit at 1 cm) with the A260/A280 and A260/A230 ratios; then converts ng/µL to nM and copies per µL from the length or sequence of your plasmid, PCR product, oligo or RNA, and lays out a serial dilution series for a qPCR standard curve.
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At a glance
| Concentration | A260 × factor × dilution / path; dsDNA 50, RNA 40, ssDNA oligo 38 ng/µL per unit |
|---|---|
| Purity ranges | A260/A280 ≈ 1.8 (DNA), ≈ 2 (RNA); ≤ 1.6 suggests contamination; A260/A230 2.0–2.2 |
| Copy number | c ÷ molar mass × 6.02214076 × 10²³ mol⁻¹ |
| Molar mass | From nucleotide elemental formulas and CIAAW 2021 atomic weights; linear or circular, sodium salt or free acid |
| Standard series | Transfer and diluent per tube, copies per reaction; CSV |
| Runs in | Your web browser; nothing is uploaded |
| Price | Free, no account needed |
What it does
How to use the DNA concentration & copy number calculator
- Measure the absorbanceEnter the A260 (and A280 and A230 if you have them), the path length in cm (1 for a standard cuvette) and any dilution factor.
- Pick the sample typeDouble-stranded DNA, single-stranded DNA or oligo, RNA, a custom factor, or protein at 280 nm.
- Convert to copiesClick “Use in copy number”, choose the molecule (plasmid, PCR product, oligo or RNA) and enter its length or paste its sequence.
- Plan the standard curveClick “Make a standard series”, set the top standard, fold dilution, number of points and tube volume.
- Save itCopy the result or download the dilution series as a CSV.
How it works
c (ng/µL) = A260 × factor × dilution / 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 = A280 × dilution / (ε × 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).
copies/µL = c (g/µL) / M (g/mol) × NA
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. NA = 6.02214076 × 10²³ mol⁻¹, exact since the 2019 redefinition of the SI (Natl Sci Rev 2020).
Worked example
A plasmid miniprep reads A260 0.75, A280 0.40, A230 0.36 in a 1 cm cuvette. Concentration = 0.75 × 50 = 37.5 ng/µL; 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 × 10⁶ g/mol (661.86 g/mol per base pair). 37.5 ng/µL = 37.5 × 10⁻⁹ g/µL ÷ 1.98559 × 10⁶ g/mol = 18.89 fmol/µL (18.89 nM), × 6.02214076 × 10²³ = 1.137 × 10¹⁰ copies/µL.
Standard series. For seven 10-fold standards from 10⁷ 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).
Frequently asked questions
How do you calculate DNA concentration from A260?
Multiply the A260 by 50 ng/µL for double-stranded DNA (40 for RNA, about 38 for single-stranded oligos) and by any dilution factor, and divide by the path length in cm. An A260 of 0.75 in a 1 cm cuvette is 37.5 ng/µL of dsDNA.
What A260/A280 ratio means my DNA is pure?
A ratio of about 1.8 is generally accepted as pure for DNA; 1.6 or below suggests protein, phenol or another contaminant absorbing near 280 nm. Pure RNA gives about 2.
What does the A260/A230 ratio tell me?
It is a second purity check: values of 2.0–2.2 are commonly expected for pure nucleic acid, so a lower value suggests a less pure sample.
Which factor should I use for oligonucleotides?
Cavaluzzi and Borer (2004) recommend 38 µg/mL per absorbance unit for single-stranded DNA of non-repetitive sequence with 40–80 % GC; they found that predictions from nearest-neighbour or base-composition extinction coefficients give concentrations 10–30 % too low for typical PCR primers. Kline and Duewer (2020) give 37–40 for single-stranded DNA.
How do I calculate copy number from ng/µL?
Divide the concentration in g/µL by the molar mass of the molecule in g/mol and multiply by Avogadro’s constant, 6.02214076 × 10²³ mol⁻¹. For 37.5 ng/µL of a 3,000 bp plasmid (sodium salt), that is 1.137 × 10¹⁰ copies/µL.
Why does the counter-ion change the copy number?
The molar mass includes the counter-ion on each phosphate. With equal base composition a base pair is 661.86 g/mol as the sodium salt and 617.90 g/mol as the free acid, a 7 % difference in copies per ng.
How do I make a qPCR standard curve dilution series?
Dilute the stock to the top standard, then make each further point by taking a fixed volume of the previous tube into diluent (10 µL into 90 µL for 10-fold steps). The series tab lists the transfer and diluent for every tube.
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