Hello everyone,

I have been trying to do qPCR for a gene of interest, but I keep getting multiple peaks on my melt curve (image attached) after the runs. At first, I thought it may have due to unspecific primers, however, I have since tried a total of three different sets of primers, and each time I get the same pattern of multiple peaks on my melt curve with no other noticeable issues. The amplification plot has no issues (image also attached) and besides the melt curve everything from the qPCR run seems to look okay.

When I initially receive the primers, I amplify and then verify them on an agarose gel and receive one single band at the expected product size. I have also since run the qPCR products on an agarose gel, and again a band appears at the correct size. However, it seems like there may be a VERY faint, tiny band (~50bp) below the correct band. I have looked on BLAST to see if there are any possible off target binding sites, and while there are some, they are all much larger (~1500bp) and unlikely to be off target binding sites and certainly not showing up on the gel. I'm just confused as to both why the melt curve is so strange, as well as why I have a very tiny band from the qPCR product but not when I run the primers on a gel alone. The fact that this has persisted across three different sets of primers has also mystified me. Thanks for any help!