• Create a new experiment on the instrument touchscreen.
a. In the home screen, touch
Open Template
4
Genotyping
4
Genotyping Post
.
b.
(Optional)
In the
Properties
tab, edit the experiment properties.
c. In the
Method
tab, set the hold temperature to 60°C with a 2 minute hold and enter the
appropriate reaction volume.
d. In the
Plate
tab, enter the dilution series information for the appropriate wells.
3.
Touch
Start Run
.
4.
When the run is complete, download the results for analysis.
5.
Unload the plate from the instrument.
CAUTION!
PHYSICAL INJURY HAZARD.
During instrument operation, the plate temperature
can reach 100°C. Allow it to cool to room temperature before handling.
Determine the optimal dye concentration
Review the dye signal data and select the dilution to use for dye calibrate.
1.
In the
Results
tab of the desktop software, select
Raw Data Plot
.
This plot displays the raw fluorescence signal of each optical filter, for individual wells.
Note:
The
Raw Data Plot
cannot be viewed on the instrument touchscreen.
2.
For each replicate population of dilutions, select the wells in the
Plate Layout
to view in the plot.
3.
Examine the raw data to identify the wells yielding signals in the 800,000 to 3,200,000 range for the
optical filter where the dye is brightest.
4.
(
Optional
) Export the raw data, then calculate the average fluorescence value for each
concentration.
5.
Select the lowest (optimal) dye concentration that falls within the acceptable signal range.
Calibrate the custom dye
Create a custom dye calibration plate
IMPORTANT!
Wear powder-free gloves while creating the dye plate.
Create a full plate of the custom dye diluted to the optimal concentration:
1.
Dilute the custom dye to the optimal concentration in buffer.
Prepare an adequate volume, using a volume range of 10–20 µL/well for a 96
‑
well plate.
2.
Pipet the appropriate volume of the diluted custom dye to the plate wells.
3.
Seal the plate.
Chapter 5
Calibrate and verify instrument performance
Calibrate custom dyes
5
QuantStudio
™
1 Plus Real-Time PCR System Installation, Use, and Maintenance Guide
67
