What this calculator does
Copy number converts a mass concentration into a count of molecules. The chain runs from concentration in ng/µL, through the molecular weight of the fragment, to moles, and then through Avogadro constant to individual copies.
Fragment length is what drives it, and inversely. A 100 bp fragment at a given concentration contains ten times as many copies as a 1,000 bp fragment at the same concentration, because each molecule is a tenth of the mass. Quantitative work that specifies a copy number rather than a mass is doing so precisely because of this.
The formula
The molecular weight of the fragment is its length multiplied by the average weight per base or base pair: 660 daltons per base pair for double-stranded DNA, 330 per base for single-stranded DNA and 340 for RNA. Concentration divided by molecular weight gives moles, and multiplying by Avogadro constant of 6.02214076 × 10²³ gives copies.
| Term | Meaning |
|---|---|
| Copy number | The count of individual molecules, as opposed to their combined mass. |
| 660 Da/bp | The average molecular weight of a DNA base pair, used for double-stranded fragments. |
| Avogadro constant | 6.02214076 × 10²³ per mole, the number of molecules in a mole. Exact by definition since the 2019 SI redefinition. |
| Molar concentration | The intermediate step: concentration divided by molecular weight, before converting to a count. |
The inputs explained
| Field | What to enter |
|---|---|
| Concentration (ng/µL) | Concentration in ng/µL, typically from an A260 reading or a fluorometric assay. |
| Fragment length (bp (or nt)) | Fragment length in base pairs, or bases for single-stranded material. For a plasmid, use the full plasmid size rather than the insert size. |
| Molecule type | Whether the material is double-stranded DNA, single-stranded DNA or RNA, which sets the weight per base. |
| Sample volume (µL) | Sample volume in microlitres, for the total copies figure. |
When to use it
Preparing a qPCR standard curve
Standards are specified in copies rather than mass, so a dilution series has to start from a known copy number in the stock.
Setting up digital PCR
Digital PCR partitions a sample so that most partitions hold either zero or one target molecule, which requires knowing the copy number going in.
Checking a plasmid prep
Converting concentration to copies gives a sense of molar amount, which is what matters for transformation and ligation rather than mass.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
How does fragment length change copy number?
The same concentration across a range of fragment lengths.
| Fragment length | Copies per µL | Total copies in sample | Molecular weight used |
|---|---|---|---|
| 100 bp | 91,244,556,970 copies/µL | 4,562,227,848,000 copies | 660 Da per bp |
| 500 bp | 18,248,911,390 copies/µL | 912,445,569,700 copies | 660 Da per bp |
| 1,000 bp | 9,124,455,697 copies/µL | 456,222,784,800 copies | 660 Da per bp |
| 5,000 bp | 1,824,891,139 copies/µL | 91,244,556,970 copies | 660 Da per bp |
Questions
Why is a base pair 660 daltons?
It is the average molecular weight across the four possible base pairs in double-stranded DNA, including the sugar-phosphate backbone. Single strands use about 330 per base, half the figure, since only one strand is present.
What length do I use for a plasmid?
The total plasmid size in base pairs, not the insert. A circular plasmid is one molecule and its mass is determined by its full length, so using the insert size alone would badly overestimate the copy number.
Why does copy number matter more than concentration?
Because molecular reactions depend on the number of molecules, not their combined mass. Two samples at the same ng/µL contain very different numbers of molecules if their fragment lengths differ, and the reaction responds to the count.
Is this accurate enough for a standard curve?
It is the standard method and it is as accurate as the concentration measurement underneath it, which is usually the limiting factor. Fluorometric quantification is considerably more reliable than A260 for this purpose, because it is specific to double-stranded DNA rather than responding to any nucleic acid present.
To measure the concentration this starts from, see the DNA concentration calculator. For qPCR efficiency from a standard curve, see the qPCR efficiency calculator.