Two peaks in dissociation curve - Two peaks in dissociation curve (Dec/08/2008 )
Hi I am trialing real-time primers for KISS-1 mRNA. In the publication we got the primers from, they used a 70°C annealing temp. I get amplification of my cDNA samples (and not of no RT controls) at this temp. but not with a 60°C annealing temp. However, the dissociation curve shows two peaks at 87 and 90°C. I diluted the primers to optimal dilution (200 nM). When I run the product out on a gel I see a strong band at 300 bp (the expected product) and a very faint one at 270 bp. should I use these primers? Will the presence of the 270 bp band skew my real-time results or does its faintness mean that it probably doesn't come up till really late? I've tried other primers unsuccessfully; there are relatively few options for this gene.
no, that second band is definitely a problem. please verify your primer sequences and specificity, and that your template is OK. you cannot trust your realtime results if there are two bands; it's a very sensitive technique