From mass to copies
Molecular biology often needs the number of DNA molecules, not their mass: to make qPCR standards, to set up a ligation, or to calculate a transfection. The conversion uses Avogadro’s number and the average molar mass of a base pair:
Copies = mass (g) × 6.022 × 10²³ ÷ (length × average mass per base)
| Molecule | Average mass |
|---|---|
| Double-stranded DNA | about 650 g/mol per base pair |
| Single-stranded DNA | about 330 g/mol per nucleotide |
| Single-stranded RNA | about 340 g/mol per nucleotide |
Some protocols use 660 g/mol per bp for dsDNA; the difference is small.
Example
10 ng of a 5,000 bp plasmid:
Copies = 10 × 10⁻⁹ × 6.022 × 10²³ ÷ (5,000 × 650) ≈ 1.85 × 10⁹ copies
In 10 µL, that is 1.85 × 10⁸ copies per µL. To prepare a standard at 10⁶ copies per µL, dilute about 185-fold, for example by a 1:10 dilution followed by a 1:18.5 dilution.
Useful reference values
| Template | Copies in 1 ng |
|---|---|
| 100 bp PCR product | about 9.3 × 10⁹ |
| 3,000 bp plasmid | about 3.1 × 10⁸ |
| 5,000 bp plasmid | about 1.85 × 10⁸ |
| Lambda phage DNA (48.5 kb) | about 1.9 × 10⁷ |
| Human haploid genome (about 3.1 Gb) | about 300 copies (one genome weighs about 3.3 pg) |
Making a standard curve
- Measure the concentration accurately, ideally with a fluorescent dye assay rather than absorbance, which is affected by RNA and contaminants.
- Linearize plasmids if possible: supercoiled plasmids amplify less efficiently in the first cycles.
- Prepare a series of 10-fold dilutions, for example from 10⁷ down to 10¹ copies per reaction.
- Use low-binding tubes and a carrier such as tRNA or yeast RNA for very dilute standards, which can lose DNA to the tube walls.
- Prepare fresh dilutions, or store aliquots frozen to avoid repeated freeze-thaw cycles.
Limits
At very low numbers, random sampling matters: a reaction expected to hold 3 copies often contains 0, 1 or 5. Below about 10 copies per reaction, results follow Poisson statistics rather than simple dilution math.