About the DNA Copy Number Calculator
This DNA copy number calculator converts a mass of nucleic acid into the number of molecules (copies). Enter the amount, the length of the template in base pairs or nucleotides and whether it is double-stranded DNA, single-stranded DNA or RNA, and it returns the copy number, the amount in picomoles and femtomoles, the molecular weight and the copies per microlitre.
It is most often used to build absolute-quantification standard curves for qPCR and digital PCR from a plasmid or gBlock, to set up ligations and transfections at a defined molar ratio, or to check how many genome copies are in a sample. A table shows the copies per µL across a ten-fold dilution series so you can plan standards directly.
Molecular weight uses the approximate formulas published by Thermo Fisher (dsDNA 607.4 g/mol per bp + 157.9), or the popular rule of thumb of 650 g/mol per base pair. For dsDNA the two differ by about 7% (6–9% across all three types), which is comparable to the typical error of measuring DNA concentration by absorbance. Copy number is only as accurate as the concentration you enter, so quantify plasmids fluorometrically where possible.
With the default inputs, the number of copies is 198,282,110. Change any value above to recalculate instantly.
How to use the dna copy number calculator
- 1Enter the amount of DNA or RNA and its mass unit (or a concentration per µL with volume 1).
- 2Enter the template length, including the vector backbone for plasmids.
- 3Choose double-stranded DNA, single-stranded DNA or RNA and a molecular-weight method.
- 4Read the copy number and copies per µL, then use the dilution table to plan qPCR standards.
Formula and method
The number of molecules equals the number of moles times Avogadro’s constant (6.02214076 × 10²³ per mol), and moles equal mass divided by molecular weight. The molecular weight of a nucleic acid is its length times the average mass of a base pair or nucleotide, plus a small correction for the end groups; these are the approximate formulas Thermo Fisher publishes for sequences of average base composition.
Many protocols instead use 650 g/mol per base pair for dsDNA (660 is also common), 330 per nucleotide for ssDNA and 340 for RNA. Either way the formula reduces to copies ≈ ng × 6.022 × 10²³ ÷ (length × 650 × 10⁹). For linear PCR products and circular plasmids the same formula applies, since a circle has no end groups to change the mass meaningfully.
- Nₐ
- Avogadro constant, 6.02214076 × 10²³ mol⁻¹
- MW
- Molecular weight of one molecule (g/mol)
- bp / nt
- Length in base pairs (dsDNA) or nucleotides (single-stranded)
Worked examples
1 ng of a 5 kb plasmid
MW = 5,000 × 607.4 + 157.9 = 3,037,158 g/mol. 1 × 10⁻⁹ g ÷ 3,037,158 g/mol = 3.29 × 10⁻¹⁶ mol, and multiplying by 6.022 × 10²³ gives about 1.98 × 10⁸ copies.
25 ng/µL of a 200 bp PCR product, 650 g/mol rule
With 650 g/mol per base pair the amplicon weighs 130,000 g/mol. 25 ng ÷ 130,000 g/mol × 6.022 × 10²³ ≈ 1.16 × 10¹¹ copies in each microlitre.
1 µg of 1,000-nt RNA transcript in 10 µL
MW = 1,000 × 320.5 + 159 = 320,659 g/mol, so 1 µg is 3.12 pmol or about 1.88 × 10¹² molecules — consistent with Thermo Fisher’s worked example. Dissolved in 10 µL, that is 1.88 × 10¹¹ copies per µL.
Frequently asked questions
How do I calculate DNA copy number from ng?+
Copies = ng × 6.022 × 10²³ ÷ (length in bp × 650 × 10⁹). For 10 ng of a 3,000 bp plasmid this gives about 3.1 × 10⁹ copies. The calculator uses the same relationship with a more precise molecular weight if you choose.
Should I use 650 or 660 g/mol per base pair?+
Both are common rounded averages that include the sodium counter-ion; the exact value depends on base composition and salt form. Thermo Fisher’s approximate formula uses 607.4 g/mol per base pair. The choice changes copy number by under 10%, similar to the typical error of absorbance-based quantification.
Do I use the plasmid length or the insert length?+
Use the full length of the molecule whose mass you measured. For a plasmid standard that is the vector plus insert, even if your qPCR assay only amplifies a short region of the insert.
How many copies should a qPCR standard curve cover?+
A typical absolute standard curve spans five to seven ten-fold dilutions, for example 10⁷ down to 10¹ copies per reaction. Prepare dilutions fresh in carrier-containing buffer, because very dilute DNA sticks to plastic.
Does the formula work for genomic DNA?+
Yes, with the genome size as the length. The human haploid genome is about 3.1 × 10⁹ bp, so one copy weighs roughly 3.3 pg, and 10 ng of human DNA contains about 3,000 haploid genome copies.