PCR cleavage assay in vitro

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.


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Turquoise Ghoulover 1 year ago

I would suggest cutting the band and purify it and send for sequencing. Also, consider T7EI mismatch detection assay I am not sure which one you used for your work. Is the DNA is from a single clone expansion or you have mixed cells too? . It could possible cells were not properly transfected with plasmid.

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