Hello, I’m working with cas9 in vitro clevage using pcr prodcuts as targer. After cas9 digestion a ger no dna bands por band with size bigger than the dna target. My pcr product has the size expected when i runned as control. Any idea where are the dna band or what are the bigger ones?. I’m using heat inactivation at 95 degrees after the assay and also used proteinase k.
thanks!
Hello Borja,
I have the same problem. I’m trying the in vitro clevage with Cas9 (purchased from NEB) using pcr product as DNA template. After Cas9 digestion a have three bands; one correspondning to the PCR intact, one smaller corresponding to the biggest of the two expected cleaved bands and one bigger than the PCR (that is approxiamately of the sum of the size of the PCR and the smallest of the two expected cleaved bands).
Did you find an answer to your problem or a way to make it work properly?
Thanks,
Alexandra
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