Amplyfing Mitochondrial DNA Control Region

I have been having trouble amplifying the control region of two species. In one (a parrot) the primers are universal, in the other (a felid;black-footed cat) the primers were designed for the closely related domestic cat. In both I have experienced non-specific binding. I have gone through the process of optimization, changing Ta and [Mg] and even template concentration. Anyone have any ideas, or have had the same issues. Thank you.

Try hot starting your PCR. This can either be done by using a hot start enzyme or by adding the taq separately after the PCR reaches 80C (best if this is done in a large volume like 5µl).

Also try adding 500mM betaine - in my experience betaine is the best additive to use.

Daniel Tillett
Nucleics Support