In my ddPCR assay to check my specific edits the PCR amplicon length is 100 bp, and two probes are within the amplicon. Probe 1 (labelled with FAM) is specific to mutant and Probe 2 (labelled with Hex) as a reference to give an approximate count. When edit is present I expect fluorescence from both the probes (DROP ON). Since there is 1 bp difference between WT and edited strain I want to use a 3'modified non extendible DARK probe to prevent MUTANT probe to pick up the WT sequence(cross reactivity?). My question is if the dark probe prevents polymerase from extension will this inhibit the signal from the reference (hex) in the wildtype strain?
Caroline Weydert
Hi, the dark probe should also get hydrolyzed, just like a normal probe.
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