Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

qPCR inhibition - (Mar/18/2011 )

Hi all,

Im having trouble with my qPCR assays. It seems like I'm getting a inhibitor in some of my samples when I'm producing cDNA. I use Invitrogen kits to DNase treat my samples then produce cDNA and sometimes after this the samples are fine, but other times the standard curves I make from these sample are completely off. The first few dilutions won't amplify properly and only by the 4th or 5th dilution do I get a normal sigmoidal curve. Its not due to inhibition in the original sample because I have produced cDNA from the same RNA sample and once its been fine, but the second time it wasn't. I can't work out what is causing this because I'm only adding reagents from the invitrogen kits. Does anyone have any idea of what could be going wrong?

-Lakia-

what about the concentration? ... maybe it is highly underestimated?

Regards,
p

-pDNA-