Calibrate custom dyes
Overview of custom dyes
The instrument can run assays designed with custom dyes. The following items are
defined as custom dyes:
• Dyes that are not manufactured by Thermo Fisher Scientific.
• Dyes or formulations of dyes that are not system dyes for the instrument.
Custom dye calibration workflow
Determine the optimal dye concentration for each custom dye. Use this
concentration to prepare all subsequent dye calibration plates.
Dilute the custom dye
Prepare a custom dye dilution plate (page 99)
▼
Run the dilution plate (page 100)
▼
Determine the optimal dye concentration (page 100)
▼
Calibrate the custom dye using the optimal concentration
Create a custom dye calibration plate (page 101)
▼
Add a custom dye to the instrument (page 102)
▼
Perform a custom dye calibration (page 102)
Use a dilution series to determine an optimal custom dye concentration
Custom dye dilution guidelines
Prepare a dilution series for each custom dye.
• Target several dye concentrations within a range of 100–2,000 nM.
• The custom dye for use in calibration must be attached to the 5' end of a short
DNA oligonucleotide consisting of the first two bases of the probe sequence
without a quencher at the 3' end.
• Choose a 2
‑
or 3
‑
fold difference in dilution points.
• Dispense 10–20 µL per well for 96
‑
well plates.
• Dispense 5 µL per well for 384-well plates.
Chapter 7
Calibrate and verify instrument performance
Calibrate custom dyes
7
98
QuantStudio
™
6 Pro and 7 Pro Real-Time PCR Systems User Guide
