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Dissociation curve vs Gel image - Which one to trust? (Apr/24/2007 )

Hi All,

I ran a SyBr Green qPCR lately and found that only one peak appeared in the dissociation curve (indicating the PCR reaction was specific), however, it showed one extra weak band right below the correct band. I am not too sure if the primer sets can be considered reliable? Wrote to technical support, but not helpful at all. Thanks for any suggestions.

-keentolearn-

In my opinion, the gel tells the truth. Melting curves are only helpful to control a qPCR, that was already optimized.
You'll get a definite answer, if you cut both bands from the gel, purifiy them seperately and send them for sequencing...

But I would try further optimizing the PCR conditions until the second badn disappears.

K.

-kr├╝melmonster-