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.

Dear Anna,

Greetings , During my Ph D in Germany we have used the following methods

The technique involves labeling of one of the two primers of a PCR reaction with one of

the two coloured fluorescent tags (blue or yellow). The samples were mixed with 2μl internal size

standard, denatured at 90°C for 2 min and chilled on ice. The resulting PCR product(s) are

separated on polacrylamide gels with the help of the automated laser fluorescence (ALF) express

DNA sequencer. Fragment analysis was carried out on (ALF) express sequencers (Amersham

Biosciences, Freiburg, Germany) using short gel cassettes. Each sample was loaded on a

polyacrylamid gel. Denaturing 6% polyacrylamide gels, 0.35 mm thick, were prepared. The gels

were run in 1 X Tris-borate-EDTA (TBE) buffer (pH 8.0) with 600 V, 50 mA and 50 W with 2

mW laser power and a sampling interval of 1,00 sec. The gels were reused four to five times. Ineach laser, fragments with known size were included as standards. Microsatellite fragment sizes

were calculated using the computer program Fragment Analyser Version 1.02 ‘Amersham

Bioscience, Freiburg, Germany’, which was added to each lane in the loading buffer by comparison with the internal size standards (Röder et al., 1998). (Appendix Fig. 1).