I'm doing qPCR targeting 16s rRNA with the primers 337F and 518R.

The standard curve looks fine. The problem is the melting peak of the environmental samples of unknown bacterial composition. They are not sharp and not significant. It seems the samples contain something else and for 16s it shouldn't be like that as the target sequence must be conserved and frequent as far as I know!

I will highly appreciate if you could share your ideas in this case.

Thanks.

P.S: the attached picture is the melt curve and the sharp peaks are for the standards.

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