FAQs
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FAQs
Browse our frequently asked technical questions by selecting a product below.
What troubleshooting recommendations are there if no signal is detected?
We recommend optimising the reaction by applying the following:
- Increase the concentration of the probe. Keep in mind that adding too much probe can slow the PCR reaction as the processivity of the polymerase goes down when the it cleaves the probe. However, doubling the probe concentration should not cause any significant effects. Applying this should increase the fluorescence signal enough to be detected and be higher than the ROX level, if ROX is being used. We advise a titration of probe (10% more, 20% more, etc.) to find a concentration where the probe signal is high enough to be detected or is above the ROX signal, without inhibiting the PCR reaction.
- Different annealing temperatures (55-67°C) could also be tested. As this is a probe-based assay, an optimisation round will be required for each probe/primer set. To help with testing, 1x SYBRTM dye could be added to the mix so that a melt analysis can be carried out after the PCR. This helps determine if a product was obtained. If no product is obtained, this could mean the probe didn’t anneal properly therefore optimisation of the probe may be required.
