I wanna ask about taqman assay designing,
i am designing a taqman assay, I used primer3plus software to design the primers and probes
but I want to test the specificity of the assay that it will detect only my target
when i use NCBI primer blast, there is only a space to detect primers without the probe.
so is there any other method to put the primers and probe and test the assay?
thanks in advance
Hi Marina,
You can test the primers with NCBI primer BLAST, as you already do, and the probe with the default Nucleotide BLAST. Generally your assay will be specific if the primers are specific. It is important, however, to check specificity of the probe alone as well, since any off-target interactions will reduce efficiency of your assay.
Best,
Eleni
Hi Marina,
It should not be a big problem.
If your primers are specific, you will amplify only your target during realtime q-PCR. Just to keep in mind that both the primers bind only on your target region. Regarding the probe, you should find a specific region inside your target sequence. If it is difficult for you to find a specific region for the probe, you can try increasing a bit your target sequence or find another region inside the same gene/ exon. If you maintain all these, there should not be a lot to worry about, as taqman chemistry is highly specific.
Best wishes.
Using NCBI tools should be helpful. Moreover, testing your TaqMan and perform a melting courve is the best way to check reliability/efficacy
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