Observation
Suggestion
Signal fluctuates during run
Check lamp, replace if necessary. See
Changing the Lamp
.
Verify that the raw (multicomponent) fluorescence signal is below the
recommended maximum of 35000 and above the recommended
minimum of 3000 RFU. If necessary, adjust the
Filter Gain Settings
to
bring the signal into the recommended range.
If ROX is being used as a reference dye, the final ROX concentration in the
reaction should be approximately 30 nM. If the dye SYBR
®
Green I is used
as the reporter, ensure that the dye is diluted appropriately before addition
to the reaction mix.
Verify that the instrument is not subjected to vibrations from an external
source during the run, such as from another instrument on the same
bench. Also verify that the line power provided to the instrument is within
the required specifications of 100–120 volts AC (+/–10%), 50/60 Hz,
10 A for a 110V system or 200–240 volts AC (+/–10%), 50/60 Hz, 5 A
for a 230V system. If consistent power cannot be maintained by the local
power supply, it may be necessary to use a Line Conditioner. An example
Line Conditioner that is known to work well is the POWERVAR ABC700-
11MED.
Ensure the instrument is not located near a window where the back of the
instrument is exposed to direct sunlight. If this is the case, the window
should be blocked or the instrument should be relocated.
Unexpected results in one sample
Check the sample container for contaminating material.
Check optical clarity of sample container cap.
Ensure the liquid inside that sample tube did not evaporate during the run,
which would indicate a vapor leak in that particular sample. Vapor leaks
may occur if the sample was not sealed properly or incorrect plasticware
was used.
Clean the thermal block as outlined in the section
Maintaining the
Instrument
.
Decreased volume in sample containers at
end of run
Sample containers are not vapor tight. Ensure caps are tightly sealed and
containers are not malformed. Also ensure that proper plasticware was
used.
Excess condensation in reaction tubes
When using single tubes or strip tubes, install the Perfect Fit frame on the
thermal block to optimize contact between the sample tubes and the
heated lid.
No amplification plots are visible in the
Results screen
The wrong data collection point may have been assigned to the
amplification plots (e.g. the dissociation curve collection point). Reset the
data collection point for the amplification plots on the
Analysis
Selection/Setup
screen.
Ensure that a data collection point was specified on the
Thermal Profile
Setup
screen. Without a data collection point, no data is collected and
analysis is impossible.
If you selected dRn data when a reference dye was not defined for the
experiment, you will not see any amplification plots on the Results screen.
In this case, there will normally be an error message under the plot stating
“None of the wells selected have a reference dye selected.” Change the
Fluorescence to dR and the amplification plots will be visible.
Mx3000P and Mx3005P QPCR Systems Setup and User’s Guide
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