Optical (dye) calibration
Updates the optical calibration files (background, well uniformity and pure-dye spectra) and confirms the system then quantifies a reference DNA series correctly.
Why this is checked
Each dye emits light over a band of wavelengths that overlaps neighbouring channels. The instrument separates them using pure-dye spectra stored during calibration. Background and uniformity calibrations correct for plate and block fluorescence and for differences in light reaching each well. Out-of-date calibrations cause noisy curves, crosstalk and Cq errors.
Typically every 6–12 months, after lamp/LED or optics service, and when a new dye is introduced, as the manufacturer and your SOP state.
Learn how the qPCR worksStandards & tools
- Manufacturer's background calibration plate (buffer only)
- Region-of-interest / uniformity calibration plate
- Pure-dye calibration plates for each dye used (for example FAM, VIC/HEX, ROX, Cy5)
- Reference DNA (or verification plate) for a 5-point 10-fold dilution series
Prerequisites
- Block temperature verified
- Calibration plates within expiry, thawed and centrifuged as instructed, protected from light
- Block clean (a high background reading shows contamination)
Step by step
- 1Run the background calibration with the background plate and check no well is flagged as high.
- 2Run the ROI/uniformity calibration as the software directs.
- 3Run each pure-dye plate and accept the spectra only if the software reports them valid.
- 4Run a verification plate: a 5-level, 10-fold dilution series of reference DNA in triplicate plus NTCs.
- 5Fit Cq against log quantity; calculate slope, efficiency and r².
- 6Check that NTCs do not amplify and replicate Cq values agree.
Example observation table
| log10 copies | Mean Cq | Replicate SD (Cq) |
|---|---|---|
| 5 | 15.21 | 0.08 |
| 4 | 18.58 | 0.06 |
| 3 | 21.90 | 0.09 |
| 2 | 25.27 | 0.12 |
| 1 | 28.61 | 0.21 |
| NTC | No Cq | — |
How to calculate the result
- 1
Linear fit of mean Cq against log10 copies: slope = −3.349, intercept = 31.96, r² = 0.9999
- 2Efficiency = (10^(1/3.349) − 1) × 100 = (1.9888 − 1) × 100 = 98.9%
- 3
Replicate SD ≤ 0.21 cycles at all levels; NTCs show no amplification
Pass if
- All calibration steps accepted by the software (no failed wells or invalid spectra)
- Verification efficiency typically 90–110% with r² ≥ 0.98 (or as set in your SOP)
- Replicate Cq SD typically ≤ 0.3 cycles above the lowest level
- No amplification in NTCs
What to do
- Repeat the failed calibration with a fresh plate.
- If background is high in some wells, clean the block and repeat.
- If the light source is weak, replace it and recalibrate.
- Do not use the instrument for GMP assays until calibration and verification pass.
Other qPCR calibrations
LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.