double melt curves on rtPCR of DNA samples - (Oct/17/2007 )

Hi, I am having problems with one of my standards on realtime PCR (Biorad icycler with sybr green), when i run my DNA samples, i am getting a primer dimer melt curve, however on certain samples, which i assume to contain a low concentration of DNA, i get a mini product curve and a smaller PD curve. I have been advised there is a way of adding a step to the protocol allowing me to take a picture at a temperature between the the two curves, however when trying to do this i'm still not sure of the results, and obviously there is no way of getting an accurate result from this, as two melt curves will always be seen. I read a forum early from chris_sylim02, and wondered if a solution was found.
Thank you,
Katie

cant find the post you are referring to.

What the temp thing means is that you do your acquisition during cycling at a temperature between the primer dimer band and the appropriate product, 81C should suffice for most situations. No, this does not change the melt curve (if there are PD there are PD...) however I am not clear as to the comment that these are not accurate results... the amplification efficiency (when read at 81C) does not have anything to do with the melt curve process. you see primer dimers and your specific product in the melt curve, but you are only measuring the specific product during the amplification so the PD should not affect the results (other than potentially slightly lowering the efficiency due to competition for primer binding) If the problem is confirming specificity because there are two peaks in the melt curve then you run a gel. I guess I think you do get accurate results from this method and if I have misunderstood your concerns please post back.

HTH and good luck...