two peaks in melting curves, primer-dimer?, contamination? - (Dec/05/2005 )
Hi, I´m checking some primers. I got 2 peaks in all of them, one at 67-73ºC (depends on the pair primers) and another one at 90 ºC (in all of them), even in the water. I runned a gel and there is a specific band but also a difused band smaller than 100 bp. So in the water, I have only the 90ºC peak in the melting curve. It is very strange but a day before, I carried out a real time PCR with one of the primers and I didn´t get the 90ºC peak. But for the recent experiment (with several primers pair) I did new diluitons of the primers, I used the same water for the dilutions of all primers, and also for the PCR. I think that can be contaminated water. Although I got a peak of 90ºC in the water, it is not enough signal in the water to get a Ct value, in fact, is very low the signal.
What do you think?
A peak of 90ºC in a melting curve, Could be primer dimer? It is too high?
It is possible that each primer-dimer (for different pairs primers, I mean) has the same peak in the melting curve?
thanks a lot
From what I can derive, sounds like you need to get new water. Can you attach a picture of your results to look at?