Did you run the reactions on a gel?

The amplification you are seeing may be primer

dimers, which will have a melting peak. You might be using too much primer.

*Then I think that your PCR product specificity is compromised (ie: your shoulder
is not primer dimers but rather a non-specific or misprimed amplimer) This means
that you will have to change your PCR conditions to get a single product of the
expected Tm/size. Usually increasing annealing temperature, changing Mg++
concentration or addatives like DMSO are used to get better specificity

*if you are ever concerned about gDNA contamination, you can always run a control with
gDNA from your cells and see where the peak falls...that way you would know for sure

I have found with one of my amplicons that 'shoulders' disappear when I add a bit
less primer. I think this would correlate with the shoulders increasing if you add too little
cDNA? it is all about the proportion of primer to amplicon and how it affects the efficiency..

The second peak is most likely amplification of primer-dimers. This is why you see an
increase when you dilute your cDNA and you see a peak in your NTC. The way to avoid this
is to ensure you are using a large enough amount of template cDNA: con poco cdna
aumento la probabilid de formar dimmer primers

*I also think it's because of primer dimers. And I think you can see it on the gel (lower
band). Try to use less primers and more DNA

*its possible to have primer dimers when there is no template or less template.

*adhesive seal can have dna contamination, use autoclavable strips

*I was having the same problem with primer dimers, and I followed ocean
recomendation in using DMSO, and it really worked!! For me it worked the best at
5%DMSO.

*Too much magnesium can lead to more primer diimer.

*mucho primer hace q facilite el aniling entre ellos dando amplificaciones

extranas y q pueda ver contaminacion.

*Raising the primer concentration does not therefore cause an increase in the effective concentration of
the primers. Low primer concentration generally ensures cleaner product and lower background.

*However, to amplify short PCR target sequences, careful calculation of the optimum primer
concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR
product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a
larger target fragment. In order to generate the required number of PCR product molecules, a greater
number of primers may be needed. Therefore, concentration of primers higher than 1μM may be
necessary, and desirable, for short target sequences.