Techne PrimeQ - Manual (Page 177)

177
5.1 Troubleshooting
ISSUE CAUSE SOLUTION
No display on LCD
screen. No power to instrument. Check power supply is connected.
Fuse blown in plug. Check fuse and change if appropriate.
No amplification. Incorrect reaction conditions. Optimize assay and run agarose gel to
check PCR
Reaction component not
added. Check correct reagents were added.
Incorrect primer or probe
sequence. Redesign primers or probe.
No template added to reaction
or degraded template present. Repeat assay.
Reaction inhibitor present in
template. Ensure that the template is <10% of the
assay volume. Purify template further.
Sequence not present in
sample. To differentiate between true negative
results and a false negative e.g. reaction
component missing, always include a
positive control.
Dye has been exposed to light
and bleached. Do not expose components containing a
dye to light. Do not freeze-thaw
repeatedly.
Wrong filter cartridge used
during data collection. Repeat assay with correct filter cartridge
for the dye selected.
NTC shows
amplification. Amplicon or template
contamination of one of the
reagents. Repeat assay with fresh reagents.
Large standard
deviation between
replicates. Inaccurate pipetting. Use a master mix for the reagents.
Use a positive displacement pipette.
High fluorescence in
one well. Sample splashed on to plate
seal. Remove plate and inspect, if droplets on
the film are easily visible, exclude the
data from analysis.
Extra master mix added in
error. Check the volume in the well.
Sudden drop in
fluorescence. Splash/droplet of master mix
running down the well. Centrifuge the plate briefly before
thermal cycling.