FAQ

De novo assay designs often require optimisation and no signal generation may be an indication of inappropriate oligo design. However, failure to amplify may also be due to an oversight in the reaction preparation, particularly if the assay has performed well in the past. To pinpoint the problem component, review the topics below:

Tip: Try to avoid designing a probe with a Guanosine at the 5' end as G may quench some of the fluorophore emission and decrease signal generation.

1. Were all the master mix components added appropriately? If repeated, does the assay continue to fail?
2. Are the sequences of the probes and primers on the tube labels and Certificate of Analysis correct? Are the fluorophore and quencher modifications correct?
3. When you analyse the primer set with SYBR Green chemistry or run the failed qPCR reaction products on an agarose gel, do you get the correct amplification product (T_M and size)? If so, then reviewing the probe sequence may help.  Consider the following factors in your current probe design:
   • Probes longer than 30 bases have insufficient quenching and are frequently non-functional. This length limitation of probe design can be overcome with the use of our Double Quenched BHQnova™ probe. This probe format incorporates an internal Nova quencher modification between bases 9 and 10 of the probe sequence in addition to the 3’ terminal BHQ™ modification. As a result, these probes are very efficiently quenched even in longer probe sequences, allowing greater flexibility of design without sacrificing performance.
   • Is the melting temperature ( T_M) of the probe near the recommended 68 °C? If the probe T_M is too low, then the probe will not hybridise well resulting in poor signal generation.