Melting Curve PCR-Real Time

[germagno86]

Hi everybody.I did a realtimePCR for construct the standard curve.And all was perfect except for some big detail, when I saw the melting curve I noted a curve with a Tm of 80.19ºC for the samples but when I check the negatives I have saw a great peak with a Tm of 79.74ºC so I have to conclude that I have primer-dimers and only primer-dimers....the peak I saw at 80.19ºC in the samples seems not to be PCR product, it seems to be primer-dimers...Is it that correct?

[debatosh]

[QUOTE=germagno86;441088]Hi everybody.I did a realtimePCR for construct the standard curve.And all was perfect except for some big detail, when I saw the melting curve I noted a curve with a Tm of 80.19ºC for the samples but when I check the negatives I have saw a great peak with a Tm of 79.74ºC so I have to conclude that I have primer-dimers and only primer-dimers....the peak I saw at 80.19ºC in the samples seems not to be PCR product, it seems to be primer-dimers...Is it that correct?[/QUOTE

your inference is correct and u should check it by running te q-pcr product on an agarose gel and the band should come below 100bp mark and even in blank reactions as you found out. do the ct values differ from each other very much?
try ordering a new set of primers or maybe try optimizing one you are having now.........