Hi all!

I have this doubte because I´m trying to quantify a gene from a metagenomic sample ( I use degenerate primers). I use Brilliant II SYBR® Green QPCR Master Mix , and the Agilent Mx3000P. 95ºC 10 min, 40 cycles of 95ºC 30 ss, 56ºC 30ss, 72ºC 30 ss. and the dissociation curve from 55ºC to 90ºC)

At first, I have too much fluorescence in the first cycles and very poor amplification, so I decided to low down template concentration (gDNA from 25 ng to 10 ng), it worked (background fluorscence went down). But, at the same time a new peak appeared in the dissociation curve at 74ºC. Does these peak could be unspecific amplification or dimer primers?

Trying to find and answer I saw that the setting in the machine I m using, is that acquisition fluorescence is at the end of the melting time, is that correct?

I read some articles where make signal adcquisition after elongation and add 10ss at 83ºC is it correct to do that?

Or maybe to change signal adcquisition at the end of the elongation?