CALCULATE WITH CONFIDENCE

Restriction Cloning & Plasmids

Calibrated Plasmid Copy Number per Cell Workbench

Invert separate entered plasmid-target and chromosomal-reference qPCR calibration lines, then normalize their quantities by an entered reference-locus copies-per-cell convention.

Biology · experimental measurements

Require both assay intercepts and slopes so a Cq difference is not silently treated as a plasmid-per-cell measurement without calibration.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · One-copy locusCalibrated assay quantities
Plasmid target214,449.63
Reference locus3,764.93581

The bars are the two separately inverted calibration quantities on the visitor-declared comparable scale. Their ratio is normalized by the entered reference copies per cell.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
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Calculation result

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Understand the relationship

The reasoning behind the result

Each assay is inverted separately

log10(Qi) = (Cqi−ai)/bi

Different slopes and intercepts are retained rather than cancelled by an uncalibrated ΔCq shortcut.

The quantity units must be comparable before their ratio is meaningful.

Reference copies set the cell denominator

PCN/cell = Qp/Qr × Cr

Cr is visitor-entered because ploidy, locus copy number and population context are not universal.

The result is a population-average calibrated ratio under those assumptions.

Calibration does not remove extraction bias

Plasmid topology, extraction recovery, lysis, inhibition and assay specificity can affect measured quantities differently.

The workbench cannot infer single-cell distributions or plasmid segregation from bulk qPCR.

Follow the numbers

Normalize two calibrated quantities

  1. Invert the plasmid-target line at its sample Cq.
  2. Invert the reference-locus line at its sample Cq.
  3. Divide the two calibrated quantities.
  4. Multiply by the entered reference-locus copies per cell.

The result is conditional on comparable standards, extraction and the declared reference-copy convention.

Quick guide

How to use this calculator

  1. Enter complete calibration equations whose quantity axes are genuinely comparable.
  2. Enter sample Cq values produced under the calibration's applicable run and preprocessing procedure.
  3. State the biological and ploidy basis for the reference-locus copies-per-cell value.

Calculation method

Calculation and interpretation

Require both assay intercepts and slopes so a Cq difference is not silently treated as a plasmid-per-cell measurement without calibration.

log10(Q) = (Cq−intercept)/slope for each assay; calibrated plasmid copies/cell = (Qplasmid/Qreference) × entered reference-locus copies/cell.

Worked example

Normalize two calibrated quantities

The result is conditional on comparable standards, extraction and the declared reference-copy convention.

log10(Q) = (Cq−intercept)/slope for each assay; calibrated plasmid copies/cell = (Qplasmid/Qreference) × entered reference-locus copies/cell.

Supported inputs

Precision and limits

Comparable quantity axes required

Both calibration equations must resolve to quantities on a compatible basis before division.

Reference copy number is entered

Ploidy and locus copy number are not inferred from species or sample labels.

Bulk average only

The result does not describe a single-cell distribution, segregation stability or viable-cell fraction.

No topology correction

Plasmid topology, extraction bias and assay accessibility are not corrected.

No assay validation

Specificity, inhibition, dynamic range, LOD, uncertainty and replicate policy remain external.

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