Step‑by‑step guide for calibrating thermal cyclers to recognize CAL FLUOR and Quasar dye spectra, with instrument‑specific procedures and handling instructions.
This manual explains why spectral calibration is required for real‑time qPCR instruments and how it enables accurate multiplexed assays by distinguishing overlapping fluorescent signals. It details the preparation, storage, and protection of calibration standards, as well as optional steps such as reaction‑plate centrifugation.
The document provides instrument‑specific calibration procedures for a range of platforms (ABI Prism 7700, ABI 7500, Bio‑Rad iCycler IQ/iQ5/Opticon, Cepheid SmartCycler II, Eppendorf Mastercycler ep Realplex, Roche LightCycler 480) and includes tables of recommended dye concentrations, catalogue numbers, and handling guidelines.
| Amount provided per calibration standard | 5 nmol |
|---|---|
| Recommended calibration concentration series | 25 nM, 50 nM, 100 nM, 200 nM, 300 nM |
| Storage temperature for calibration standards | -20 °C or -80 °C |
| CAL Fluor Gold 540 concentration for ABI Prism 7700 | 200 nM |
| CAL Fluor Orange 560 concentration for ABI Prism 7700 | 250 nM |
Most real‑time qPCR instruments require dye calibration to distinguish fluorescent signals, especially for multiplexed assays where overlapping signals must be resolved.
Aliquot each standard into individual microvials and store frozen at -20 °C or -80 °C, minimizing freeze‑thaw cycles.
Use a dilution series of 25 nM, 50 nM, 100 nM, 200 nM and 300 nM and select the concentration that falls within the instrument’s linear detection range.