Sample calculations for quick validation
| Concentration (ng/µL) | Length (bp) | Volume (µL) | Dilution factor | Copies per µL | Total copies |
|---|---|---|---|---|---|
| 50 | 5000 | 20 | 1 | 9.26e+09 | 1.85e+11 |
| 10 | 3000 | 50 | 10 | 3.09e+10 | 1.54e+12 |
| 2.5 | 9000 | 100 | 1 | 2.57e+08 | 2.57e+10 |
These examples assume dsDNA with 650 g/mol per bp and Avogadro’s constant 6.022×1023.
How copy number is computed
Mass-based method (copies from concentration)
Convert concentration to grams per µL, compute plasmid molecular weight, then convert grams to moles and moles to molecules.
Defaults: dsDNA weight_per_bp ≈ 650 g/mol per bp; NA = 6.02214076×1023 mol−1.
qPCR-based method (copies per cell from Ct)
Relative quantity is proportional to E−Ct. With a reference gene of known copies per cell, plasmid copies per cell can be estimated from the ratio.
Use assay-specific efficiencies where possible; a single-copy chromosomal reference makes interpretation straightforward.
Steps for accurate results
- Select a method. Use mass-based for concentration data, or qPCR-based for Ct comparisons.
- Enter inputs with units. Check concentration, plasmid length, volume, and dilution factor.
- Pick the DNA type. dsDNA is typical for plasmids; use custom weight only when justified.
- Provide replicates (qPCR). Enter comma-separated Ct values to average noise across runs.
- Click Calculate. Results appear immediately above the form under the header.
- Export your work. Use CSV for spreadsheets and PDF for sharing or archiving.
Copy number in practice
Plasmid copy number influences expression, stability, and burden. High‑copy backbones can exceed 100 copies per cell, while low‑copy systems may stay near 1–5. Knowing copies helps choose antibiotic strength, set induction levels, and compare yields across minipreps. This calculator offers two routes: a mass approach for purified DNA and a Ct approach for cellular measurements, both suitable for routine documentation. If you track copies per microliter over time, you can spot degradation after freeze‑thaws and decide when to remake stocks for consistent transformations across multiple projects.
Mass-based conversion details
Mass-based estimation converts concentration into molecules. First, convert your reading to ng/µL, then to grams per µL using 10⁻⁹. Molecular weight is approximated as length(bp) × 650 g/mol for double‑stranded DNA. Moles per µL equal mass/MW, and copies per µL equals moles × 6.022×10²³. Apply the dilution factor if you measured a diluted aliquot, then multiply by total volume for total copies.
qPCR ratio interpretation
qPCR-based estimation compares plasmid and reference targets. Relative quantity scales with E^−Ct, where E is amplification efficiency (2.0 equals 100% efficiency). The plasmid-to-reference ratio is (Eref^Ctref)/(Eplasmid^Ctplasmid). Multiply that ratio by the known reference copies per cell, commonly 1 for a single‑copy chromosomal locus in haploid bacteria, to obtain plasmid copies per cell. Enter replicate Ct values to reduce run‑to‑run noise.
Controls and uncertainty
Good inputs matter more than extra decimals. Use accurate plasmid length, including inserts and regulatory parts. For spectrophotometry, contaminants inflate ng/µL, so fluorometric DNA assays improve copy estimates. For qPCR, confirm single peaks and similar efficiencies; a difference of 0.1 in efficiency can shift ratios measurably across 20–30 cycles. Record dilution steps, because a 1:10 oversight creates a tenfold copy error.
Reporting and exporting
Exported results strengthen traceability. Store each run in the session history, then download CSV for lab notebooks, pipelines, or LIMS import. The PDF summary is useful for sharing with collaborators or attaching to batch records. For reports, include method, concentration unit, plasmid size, dilution factor, and efficiency assumptions. When comparing experiments, keep the same reference target and extraction workflow to avoid systematic bias.
Common questions
1) Which method should I choose?
Use mass-based when you have purified plasmid concentration and size. Use qPCR-based when you have Ct values for plasmid and a chromosomal reference and want copies per cell from living samples.
2) Why does dsDNA use 650 g/mol per bp?
It is a common approximation for average base composition in double‑stranded DNA. Small composition differences have minor impact compared with concentration or length errors, so 650 is practical for routine plasmid work.
3) How do I enter qPCR replicates?
Type Ct values separated by commas or spaces, such as 18.2, 18.1, 18.3. The calculator reports mean and standard deviation and uses the means for the ratio calculation.
4) What efficiency value should I use?
If you do not have a standard curve, start with 2.0 for both targets. For best accuracy, compute efficiency from a dilution series and enter the measured values for plasmid and reference assays.
5) How should I set the dilution factor?
Enter the fold dilution applied before measurement. A 1:10 dilution means a factor of 10, because the original sample is ten times more concentrated than the measured aliquot.
6) Do the downloads include my data?
Yes. CSV exports your session history as rows you can open in spreadsheets. PDF exports a short report of the latest calculation plus a snapshot of recent history for sharing or archiving.