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SYBR melting curve vs RT-PCR gel - (Nov/04/2009 )


I was trying to validated a custom designed taqman probe -- I ran a SYBR green melting curve and independently ran a RT-PCR reaction using the same RNA then ran the product on a gel. The interesting thing is, I got only 1 peak in the SYBR melting curve, which usually means that there's only 1 PCR product, but I got 3 distinct bands on my gel! Other than my <100 bp expected PCR product, I also saw a 200 bp and a 600 bp band. Any idea what's going on here?



Did you use the same PCR conditions for both?

Usually it's not needed to run a separate reaction to put on gel, you can analyse your samples on real-time cycler and then load the same reaction right onto gel. SYBR doesn't interfere with EtBr staining. That way you know for sure, that what you see on the gel and on the melting matches.