Techne PrimeQ - Manual (Page 67)

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change between these options. If the user is creating a new protocol and the instrument is
not connected, it is not possible to select filter cartridges (the drop-down box is greyed-out).
The user will be prompted to choose the filters on connection to the instrument or when
trying to run the program.
• Light Intensity: Choose low, medium or high depending on the type of fluorophore in use
and its concentration. Default: Medium.
• Integration: This is the length of time during which fluorescent data is collected for each
well. This may need to be increased for weaker dyes or where there is a high level of
background fluorescence present in the chemistry. Increasing the integration time in the
latter case can improve the signal-to-noise ratio. The integration time can be changed from
250ms for weak dyes down to 50ms for stronger fluorophores (the default is set at 150ms).
• Plot Colour: The colour displayed will depend on the selected filter cartridge and will default
to the colour which was chosen for that particular filter during installation. Double-click to
bring up other options.
• Remove Read: As the name suggests.
• Add Read: If another dye read is to be added (up to four dye reads can be added per step),
click here and repeat the process choosing the filters appropriate to the second dye. The
reads will be tabbed at the top of the settings box allowing the user to easily navigate
between them.
Repeat this procedure, adding reads where appropriate. The temperature profile graph will depict
the read points in the assigned colour.
Separate reads appear as individual tabs in the settings box; simply click to view or
change the parameters.
Any stages assigned a read will be shown in red in the program file view and the read will be
represented on the thermal profile plot by a circle in the chosen plot colour.
3.4.3.6 Setting up a ramp read stage
The Add Ramp button allows setup of a dissociation (melt) curve stage whereby the temperature
is raised in small increments between a defined range of temperatures; typically from the primer
annealing temperature up to the system denaturation temperature of 95°C.
By taking fluorescence readings at each temperature increment and then plotting a dissociation
curve, the point at which the dsDNA template melts into two strands can be identified. This
procedure is useful in product identification (the exact dissociation temperature is a characteristic
of the GC content, length etc.) and is commonly performed at the end of a PCR run where an
intercalating dye is used, for confirmation that the correct product has been amplified.