Rotor-Gene Q MDx CE User Manual 02/2022
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To perform Optical Denature Cycling, you will need:
A preamplified PCR product to place in position 1 of the rotor. This sample should contain the
same PCR product as the samples of interest and a detection chemistry for monitoring PCR
product dissociation.
An optical denature profile. A new profile can be created or an existing profile can be edited
(see details hereafter).
An Optical Denature Cycle appears almost identical to other cycles. The principal differences are
the melt step automatically inserted at the beginning of the profile, and the sharp profile of the
denature step during cycling. The Optical Denature Cycle does not require defined hold times as
the dissociation of the product is monitored at each cycle.
To perform this technique, the following information about the run is required:
The initial denaturation temperature. This is the same temperature as the Denature step in a
standard cycling profile.
The tube position of the PCR sample that will produce a melt curve on the green channel.
An Optical Denature Cycling profile must be defined.
Create a new Optical Denature Cycle as follows.
1.
Open the
Edit Profile
window. Then click on
New
. In the window that appears, click the
Insert
after
button and select
New Cycling
from the menu. Select one of the temperature steps by
clicking on the graph. In the drop-down menu, change from
Timed Step
to
Optical Denature
. A
default profile containing a Denature step and an Optical Denature Cycle step will appear.
The ramped region at the beginning of the run represents the calibration process. The green
dots represent the acquisitions taken each cycle during heating. The blue dots represent the
acquisition at the end of the anneal step at 60°C. Note that while the profile shows each step
with the same denature temperature, this may not be the case. If the sample requires slightly
longer to melt towards the end of the run, the optical denature process waits for the melt
according to the fluorescent data, and not according to time. For this reason, the temperature
trace may vary for each cycle.
2.
Click on the first half of the graph with the Optical Denature symbol
. The
Calibration
Settings
information appears on the left of the screen.