I am recently learning how to perform qPCR and have encountered some issues. I made standards from plasmid DNA and have also some cDNA samples. In both cases, when I run agarose electrophoresis, I get only one band of expected size from my qPCR. The melting curve is a different story.
I see two peaks in my standards but they always look the same despite concentration or it being a different run. The problem is that my samples have odd graphs with more peaks. I could say maybe its the sample issue but it's not consistent between technical replications and that's the most confusing part.
I have noticed that my GOI has low expression. However, the pattern in my standard curve doesn't change in the low concentration points so I don't think that's the issue.
Its not that I am amplifying something else in the samples, because the electrophoresis shows 1 band... or could it be its two bands of the same size so I cannot tell? but then what about the technical replications melting curves not looking the same? I think its primer dimer but then why does it not happen in my standards? does plasmid DNA prevent primer dimers o.O my water is clean.
I am going to try making new primers but I still want to understand what happened. Can someone help me?
Dear Emi,
Sometimes the problem is with the chemistry of Master Mix. Try to use diferent Master Mix purchased from diferent company. This should help.
Hi
the melting curve refers to the temperature at which the DNA will melt. if there are two curves, it means that you have two types of molecules. in your case, I will say that for one sample you have a normal target and a mutated target. take a look at this old paper (https://www.ncbi.nlm.nih.gov/pubmed/9056205). Melting curves analysis was a good way some time ago to genotype individuals in human studies.
Can you sequence your samples?
fred
Thank you both for your time and help, I will check both options and read the paper!
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