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Convert DNA or RNA mass to copy number and pmol, work back from copies to mass, and plan a qPCR standard curve dilution series. For plasmids, PCR products, gBlocks, oligos and RNA transcripts.
Defaults: 660 g/mol per bp for dsDNA and 330 per nt for ssDNA and RNA (sources below). Some calculators use 617.96 g/mol per bp (NEB, QIAGEN) or 650; enter that here to match.
Type 1e6, 1×10^6 or 1,000,000.
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The calculator turns mass into moles with an average molar mass per base pair or nucleotide, then multiplies by the Avogadro constant. The same steps run in reverse to go from copies to mass.
| Template | Value | Source |
|---|---|---|
| dsDNAPlasmid, PCR product, gBlock, genomic DNA | 660 g/mol per bp | Applied Biosystems (Thermo Fisher Scientific), Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR; IDT, Calculations: converting from nanograms to copy number |
| ssDNAOligo, single-stranded DNA | 330 g/mol per nt | IDT, same article (half the dsDNA value) |
| RNAIn vitro transcript, ssRNA | 330 g/mol per nt | Same value as the instruction manual of our mRNA Capping Kit (BP-04). Published values run from about 320 to 340: Thermo Fisher Scientific’s DNA and RNA Molecular Weights and Conversions gives (nt × 320.5) + 159 and an average ribonucleotide monophosphate of 339.5 g/mol |
| Avogadro constant | 6.02214076 × 1023 mol−1 | Exact value in the SI since 2019 (BIPM) |
These are averages for a typical base composition. Reference tables differ by a few percent depending on whether they count the sodium counter-ions and the end groups: NEB and QIAGEN use 617.96 g/mol per bp plus 36.04 g/mol for the ends, and Thermo Fisher’s table gives (bp × 607.4) + 157.9. Against 660 g/mol per bp, these give about 7–9% more copies for the same mass. Pick one value and keep it for the whole project. For oligos, the synthesis report gives the exact molecular weight and amount, which are more accurate than any average.
The planner converts the stock to copies/µL, works out the overall dilution to the highest standard (S1), then makes each lower standard by transferring the volume per standard ÷ the dilution factor into diluent. If the stock volume needed for S1 is below the smallest volume you pipette, it adds 1:100 or 1:10 pre-dilutions first, as in the Applied Biosystems protocol. After the run, plot Cq against log10 copies to get the slope and amplification efficiency; our guide to qPCR results analysis covers reading the standard curve, Ct values and melt curves.
1 ng of a 3 kb plasmid ≈ 3.04 × 108 copies
The plasmid is 3,000 bp. The goal is six 10-fold standards from 107 copies/µL, 100 µL each, with 2 µL of standard per qPCR reaction.
100 ng of a 1,000 nt RNA ≈ 1.82 × 1011 copies (0.303 pmol)
Divide the mass by the molar mass of one molecule, then multiply by the Avogadro constant: copies = mass (ng) × 6.022 × 1023 ÷ (length (bp) × 660 g/mol × 109). For example, 1 ng of a 3,000 bp plasmid is 1 × 6.022 × 1023 ÷ (3,000 × 660 × 109) = 3.04 × 108 copies. For ssDNA and RNA, use 330 g/mol per nt.
All of them appear in reference tables. The differences come from whether the value counts the sodium counter-ions and the end groups. 660 g/mol per bp is the value used in the Applied Biosystems standard curve protocol and by IDT, so it is the default here. NEB and QIAGEN calculators use 617.96 g/mol per bp plus 36.04 g/mol for the ends, which gives about 7% more copies for the same mass. The gap is small next to pipetting and quantification error; what matters is using the same value throughout a project. You can change it under “Average molar mass”.
Use the whole plasmid: vector plus insert. You weigh the entire molecule, so its full length sets the molar mass, even though the qPCR assay only amplifies the insert. Some studies have found that circular, supercoiled plasmid standards amplify less efficiently than linear ones, so many labs linearize the plasmid with a restriction enzyme that cuts outside the target before making standards. The plasmid calibrators in our Plasmid DNA Residue Detection Kit (qPCR) are also supplied linearized.
About 300. A haploid human genome is roughly 3.1 × 109 bp, which at 660 g/mol per bp weighs about 3.4 pg, so 1 ng holds about 290–300 haploid genome copies. For a single-copy gene that is also the number of gene copies. To check, select dsDNA, enter a length of 3.1 Gb and an amount of 1 ng.
Not directly. Plasmid copy number per cell is the ratio of plasmid copies to chromosome copies in the same sample, usually measured by qPCR with one assay on the plasmid and one on a single-copy chromosomal gene. Use this calculator to prepare absolute standards for both targets, quantify each in the same DNA extract, then divide plasmid copies by chromosomal gene copies.
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