Rotor-Gene Q MDx CE User Manual 02/2022
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8.
Set HRM run conditions. Modify the conditions to suit the amplicon. For the first set of
experiments allow for a wide melt domain. Use the theoretical
T
m
as a guide to a suitable
range. Once you have determined where the product will melt, reduce the melt domain to no
greater than 10°C. Ensure that the start of the melt occurs 5°C prior to the first melt transition.
The default ramp is set to 0.1°C with a hold of 2 s at each step. The minimum ramp transition
is 0.05°C with a second hold at each step. Data is automatically acquired to the HRM
channel. Automatic Gain Optimisation is performed by default. The software will search for
the optimal gain setting so that the highest fluorescence value reported is no greater than
70 units on a scale of 100. Note that this can be increased to a maximum of 100.
9.
Optional: Set Auto-Gain Optimisation. This applies to the real-time amplification step only and
is set for the green channel. Click the
Optimize Acquiring
button (to optimize only those
channels used by a run). Optimization is best performed just before the first acquisition step,
so check the
Perform Optimization Before First Acquisition
checkbox. The recommended
background fluorescence range for intercalating dyes is between 1 and 3 Fluorescence units.
To change this setting, click the channel name to select it in the list and then click the
Edit
button.