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.
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