amplification plot raises to early - What is the explanation for this problem? (Mar/30/2011 )
We are using the StepOnePlus machine from Applied Biosystems for Real Time PCR (qPCR) using the sybr green method. In the figure I include, you see two curves for the amplification plot. The blue one is a normal amplification plot. The signal stays zero untill round 16, when it increases rapidly.
The purple line is the problem. There the curves start raising slowly already at round one. We see this happening very often. Although some wells seems to have this problem more then other wells, it occurs still randomly. Also there is no clear correlation with the primer sequence. With the same primer set, some wells may have the problem while other wells don't have it. We perform the qPCR in triplicate. In general, just one out of these three hows the problem.
Usually there are only a few wells that show the problem, but sometimes more then 80% of the wells show the problem. And in rare cases, all the wells from one primer set shows this problem.
Who can explain what is happening here?
Thanks for your reply.
I don't have an explanation, I can only tell you I wouldn't call this "a problem". This happens very often and baseline substraction fixes it, I don't think it affect your results in any way.