This is a qPCR experiment. The red and yellow melt curves are from a qPCR of an oncogene which was transfected into a host cell; the purple and the green are the control, i.e., the same qPCR assay but without the transfected oncogene (only the vector).
Hi Marconi,
looks like Primer dimers. I assume the Ct-values of the pink and green curves are much higher than those of the other ones. To be sure do an agarose gel for your products as primer dimers run cloudy at the bottom of the gel at roughly ~80bp in general.
Best regards.
Hi Soner,
The Ct-values of the pink and the green are 27.24 and 27.67 respectively; the Ct-values of the red and yellow are 23.38 and 24.61 respectively. Aren't they close to the test samples? The agarose gel is a good suggestion, I will do it. What worried me was that it seems there is a small peak of the pink sample at the same temperature of the amplicon Tm. It is possible to see two small peaks in the pink curve, am I right? This kind of curve (similar to the pink curve) was unfamiliar to me.
Thanks.
Best regards.
You have melt-peaks from two different PCR products. Try running your samples out on a gel. The low melt temperature of the green curve suggests it's possible primer dimers, but you can't be sure unless you run a gel.
You are correct that the "double peak" in the pink sample means you have two different products. My guess is that you have contamination of your oncogene DNA in your pink sample. Again, run out some of your product on a gel. qPCR is very, very sensitive and will pick up small amounts of contamination much more easily that normal end point PCR.
Let us know what your gel looks like and we can help you proceed.
Thank you Dr Burnette for the explanations, they were very helpful. I will run the gel and let you know.
Thank you all for the support.
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