I'm trying to perform an in vitro cleavage assay with Cas9 from S pyogenies. I'm using a PCR product as a target with the size of 500 bp. The problem that I have is that the Cas9 is not cutting or not detaching the cleavage product because I see the PCR product after the assay but not cleavage bands.
I used the same protocol that I use when I cleavage plasmid DNA that works very nicely. What do you think that is the problem?. I used EDTA and 65ºC denaturation after the cleavage also.