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Dye calibration
When to perform
the calibration
About the dye
calibration data
QuantStudio
6 and 7 Flex Real-Time PCR Systems v1.6 Maintenance and Administration Guide
™
10. Unload the calibration plate or array card:
a. After the calibration, tap
card.
Note: If the instrument does not eject the plate or array card, remove it as
explained in "Troubleshooting".
b. Remove the calibration plate or array card from the instrument tray and
discard it.
WARNING! PHYSICAL INJURY HAZARD. During instrument
operation, the plate or array card temperature can reach 100°C. Allow
the consumable to cool to room temperature before removing.
c. Tap
on the instrument touchscreen to close the instrument tray.
11. From the Uniformity Calibration screen, click Finish to complete the calibration,
then click Yes when prompted to save the results.
12. (Optional) Click Print Report in the upper right corner of the screen to print a
summary of the calibration results for your records.
The software uses the calibration data collected from the dye standards to characterize
and distinguish the individual contribution of each dye in the total fluorescence
collected by the instrument. After each run, the instrument software receives data in
the form of a raw spectra signal for each reading. The software determines the
contribution of the fluorescent dyes used in each reaction by comparing the raw
spectra to the pure spectra calibration data. When you save an experiment after
analysis, the instrument software stores the pure spectra with the collected
fluorescence data for that experiment.
Perform a dye calibration once every 6 months.
IMPORTANT! Perform a background calibration before every series of dye
calibrations. Because the age and use of instrument components can affect spectra
readings, you must perform a dye calibration at least every 6 months.
The product of a dye calibration is a collection of spectral profiles that represent the
fluorescence signature of each dye standard. Each profile consists of a set of spectra
that correspond to the fluorescence collected from the wells of the calibration plate or
array card. Following the calibration of each dye, the software "extracts" a spectral
profile for each dye. The software plots the resulting data for each profile in a graph of
fluorescence versus filter.
When the software extracts the dye calibration data, it evaluates the fluorescence
signal generated by each well in terms of the collective spectra for the entire
calibration plate or array card. Dye spectra are generally acceptable if they peak
Chapter 2 Calibrate the instrument
on the touchscreen to eject the plate or array
2
Dye calibration
53
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