I have gotten gel electrophoresis results of a sample of dna which I digested with Cas9 and gRNA to test the functionality of the gRNA. The results seem a little unclear to me. All the wells which had guide show two bands, while the ones which had no guide have only one. This seems to be the result I am looking for but I am not sure. Can anyone shed some light on this?
Mateo Glumac
Hi,
this results are inconclusive. You can not claim this is successful of failed experiment because there are no clear cleavage bands.
- You should improve your PCR amount that you use for digestion at least 3 times
- Gel should be run for longer to get better separation
- Load sgRNA, Cas9 and RNP complex separately to see what background they cause
- Design your experiment like this: first well RNP complex - PCR followed by PCR without digestion in equivalent amount. If you are testing on the same PCR then you can load only one PCR product
- Products should be clean and sharp not a smear
0 votes
0 thanks
- Badges
- Science method
More Dara Eoin Meehan's questions See All
Similar questions and discussions