Thermo Scientific Applied Biosystems QuantStudio 7 Pro - Use a Dilution Series to Determine an Optimal Custom Dye Concentration

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 115)
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Run the dilution plate (page 116)
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Determine the optimal dye concentration (page 116)
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Calibrate the custom dye using the optimal concentration
Create a custom dye calibration plate (page 117)
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Add a custom dye to the instrument (page 118)
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Perform a custom dye calibration (page 118)
Use a dilution series to determine an optimal custom dye concentration
Custom dye dilution guidelines
Prepare a dilution series for each custom dye.
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Target several dye concentrations within a range of 100–2,000 nM.
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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 dierence in dilution points.
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Dispense 10–20 µL per well for 96‑well plates.
•
Dispense 5 µL per well for 384-well plates.
•
Dilute the dye in buer compatible with your Master Mix.
•
(Intercalating dyes only) Add the appropriate amount of amplified PCR product to generate
fluorescence.
Chapter 7 Calibrate and verify instrument performance
Calibrate custom dyes
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