I am running PCR on cDNA that has been reverse transcribed from mRNA extracted from the brain. I am new to PCR and am trying to optimize conditions for single cell RT-PCR. I diluted the mRNA to 1:1000 to get closer to single cell mRNA concentrations. Everything was working well when using 30 cycles, the positive bands on the gel looked nice and there was no band in the negative control. We then tried single cells. We kept everything the same except we increased to 40 cycles, and we then saw a band in the negative control. The band is the same size as our amplicon. To troubleshoot the contamination we went back to using the whole brain mRNA 1:1000 dilution. At 40 cycles the resulting DNA band is actually weaker than it was at 30 cycles. The band in the negative control is still present and actually brighter than the band in the DNA lane. It is still the same size as our expected amplicon. So I have a few questions. Does anyone have an idea as to why the DNA signal would be weaker at 40 cycles than 30, and why the contamination in the negative control would be brighter than the band in the DNA lane? And what, if anything, does it imply about the contamination that it is the same size band as the DNA band? Thanks!