Use qPCR with a standard curve of known concentrations of human gDNA as a reference. You can then use those data to easily calculate the concentration of gDNA in each sample. The melt curve does NOT tell you the concentration of your samples. Seeing multiple peaks usually means you are using primers that amplify too large of a PCR product for accurate qPCR.

Or you can use a Qubit to directly measure the concentration of DNA

Thanks for your answer. Actually, we do not need to measure the concentration of all DNA, we need to compare between the amounts of specific amplified DNA fragment.

Do you want to try running a second PCR using the first PCR reaction as a template? This might give you strong enough bands for comparison.

Tnank you for your answer. That's what I actually meant by nested PCR.

Take few amount of DNA from the gel and use it as a template in the next PCR your band will be clear and the amount of DNA will also be concentrated for sequencing etc.

My another suggestion is that, take the whole band from the gel by knife or other you can isolate this a small tip, and put in the eppendorp tube and eluate the DNA from this tube, then use the eluated DNA as a template following the same conditions of your old PCR only changing the DNA sample, the PCR will amplified that specific amplified product and your DNA will be concentrated for further purposes like sequencing etc.

Hello, Anna. A. Shmakova,

did you already fix the problem? From my experience, for small amounts of target molecules, it is the best to use droplet digital PCR (ddPCR) from BioRad. Maybe you could find some collaborators who has it?

Best regards,

Mirjana

Thank you for your answers!

Mirjana , that's a really valuable suggetion, ddPCR was one of our options.

But, finally, we managed to fix this problem by changing primers, which seem to be more specific and give a smaller amplification product. And they worked well with a qPCR.