LightCycler 480 Melting Curve Assay - (Apr/30/2012 )
We are using a multiplex (targeting 3 genes) LightCycler 2.0 assay for the identification of Campylobacter and discrimination of C. jejuni from C. coli, that we have converted to the LightCycler 480 since our lab does not have a LightCycler 2.0. We thought we had fully optimized the assay to the LC 480 considering our controls worked very well, however, there seems never to be any consistency from one run to another using the same controls. For example, one day our melting peaks look very sharp, next day jagged, next day dull instead of sharp, back to sharp,, etc. More than anything else, I believe this is a probe issue, which are using from TIB MOLBIOL (LC probes). However, I do not know the exact reason as to why the probes would behave in such a way. We have followed the exact handling and storage of the probes as recommended by the supplier. Any feedback or advice?
Jagged curves..do you use repeatedly the same plate (unused wells) by a chance?
One other idea, do you mix all the probes for storage (or are they mixed) or you add them separately? Sometimes there can be sequence-based interactions with the primers and probes, so either adding them separately to the reaction premix or denaturing the mixture before pipeting can in this case.