Real time PCR - small peaks in the melt curve - (Jul/19/2005 )

Hello. I am doing real time pcr using the soil DNA samples. I use SYBR Green I for detecting. I always got lots of small peakd in the melt curve. I used the qPCR kit from invitrogen. I tried to use the SYBR mix and ROX in the kit and DNA sample without any primers. The melting curve still had lots of small peaks which is similar to the samples. the peaks start very early, around 60oC. Any idea about that? How can I get rid of these samll peaks?

Thanks very much.

Hi there, the early peak in the melt cure are probably due primer dimers formed during the PCR. You can confirm this by running the samples on an agarose gel. To over come this, just like in normal PCR, you can increase the primer annealing temp and/or decrease primer concentration, etc.

HTH

Jeff

It should not be caused by primer dimer, because i still got the same series of small peaks at the control without primers. I think maybe it is from the buffer or something like this. Did anyone meet this situation before?

Thanks for any idea.

Primer-less reactions should definitely not give major bands. With regards to your minor peaks, what is the scale of the Y-axis on the melting curves. Perhaps these are mere fluctuations (noise), which are small and negligeable with comparison to the specific signals.

I agree with Serge

I would add, do you see the same little peaks in your H2O control wells? there is always a tiny bit of background noise