c.
Complete the New Dye dialog box, then click OK.
Field/option
Action
Name
Enter a name for the custom dye.
Wavelength
Enter the wavelength at which the dye fluoresces.
Type
Select:
• Reporter if the dye works in conjunction with a
quencher dye to report an increase of PCR product.
• Quencher if the dye suppresses the fluorescence of a
reporter dye until amplification of PCR product.
• Both if the dye reports an increase of PCR product
without the aid of a quencher dye.
d.
Click Close.
3.
In the Dye window, enter a temperature setting for the calibration. Set the
temperature to match the temperature at which you intend to collect data. For
example, the temperature for all system dyes is 60°C because data collection for
TaqMan
®
reagents occurs during the 60°C extension step of the PCR.
4.
Load the appropriate dye plate into the plate adapter, select Please check the box
when the dye calibration plate has been loaded, click Next twice, then click
START RUN to start the calibration.
5.
When the run is complete and the system ejects the plate, remove and discard the
plate or the array card.
WARNING!
PHYSICAL INJURY HAZARD. During instrument
operation, the plate can reach 100°C. Allow the plate to reach room
temperature before removing.
6.
In the Dye dialog box of the QuantStudio
™
Real-Time PCR Software, click Next.
7.
Verify the grouping of the dye spectra:
a.
In the plate layout, select the wells of the plate.
b.
Inspect the raw data. For each spectrum, verify that the peak is:
• Within the detectable range for the system.
• Free of irregular spectral peaks.
• Present at the correct filter for the dye.
Note:
Among wells containing the same dye, variations in spectral position and
peak position are caused by minor differences in the optical properties and
excitation energy between the individual wells.
8.
Verify the status of the calibration. If the calibration:
Passed—If all spectra are acceptable, finish the calibration:
a.
Click Next.
Appendix F
Calibration consumable preparation
Create a custom dye plate for calibration
F
156
QuantStudio
™
6 and 7 Flex Real-Time PCR Systems v1.6 Maintenance and Administration Guide
