Techne PrimeQ - Manual (Page 73)

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• Show all wells: The well information table will show only the details for the currently
selected sample type, e.g. standards, unless this button is clicked. Click again to change
back.
• Reset names: Allows any user-defined changes to the names to be returned to the default.
• Function buttons: Icons on the left-hand side provide easy access to basic functions
(further details in section 3.4.4.6). Click once to activate.
3.4.4.2 Sample types overview
Before setting up a real-time PCR experiment, you must consider what types of samples are to be
used in the assay. Sample types generally fall into the following categories:
• Standard (STD): Samples of known concentration are used to construct a standard curve in
order to extrapolate unknown sample concentrations.
o
Standards should always be prepared carefully since the accuracy of a
quantification assay can be no better than the accuracy of the standards.
o
Reduce the variability by using several points in the standard curve (at least three)
and when producing a dilution series, use serial dilutions no more than one order
of magnitude apart (1:10, 1:100 etc).
• Unknown (UNK): Samples containing an unknown quantity of the template being reported.
• No template control (NTC): Similar principle to a negative control but the missing component
is the template. A NTC controls for contamination and false-positives. For assays not using
an internal positive control (IPC), the calls for unknown sample can be based on a threshold
determined by the NTC. The threshold determines the minimum fluorescent signal that must
be achieved to assign a positive call to the sample.
• Negative control (NEG): Sample wells with one or more of the components of the reaction
mixture missing. Useful in end-point assays as above and in checking for contamination and
false-positives.
• Internal positive control (IPC): Particularly useful in plus/minus scoring. An IPC is a separate
PCR reaction requiring a different set of primers, probe and template. The template is
“spiked” into the PCR at a known concentration.
• Positive control (POS): Used to confirm that the PCR reaction is successful. Preferably uses
the same template and primer pair as the unknown samples. Can be useful in end-point
assays for defining the range of results.