Dear All, I' ve generated zebrafish mutant with spesific gene using sgRNA. When I did PCR for sequencing I got 2 different bands that never happpened to me before. So is that possible using same sgRNA with expected band around 500bp but I got almost half are 500bp and another half around 800bp? or is there any problem with my primer? Thank you
Yes double streand breages induced by CRISPR Cas9 are causing INDELS that can be several hundreds of bp. Not frequent but possible. I would suggest to sequence your PCR fragments to verify it.
(Mr. @Filippo) but my PCR result was little bit strange. I had 65 samples and the result was 35 having around 500bp, 26 having around 800bp and 4 having unclear or no band. Do you have any idea about that?
I have seen weird things when genotyping crispr mutants. I screen all my mutants by doing really short PCRs (~100bp) around the gRNA site. If the crispr worked I will usually see smeary pcr products in my F0s and then two distinct bands in my F1s. Sometimes these bands are smaller and sometimes they are larger. I also see some PCRs that don't work but it doesn't completely surprise me when I'm doing a bunch of PCRs if some just don't work. All that makes sense to me but I know I had at least one that just behaved weirdly. In the F1 generation I saw wt band a larger band so I assumed it was an insertion. However, when I got to the F2 generation and seperated out wt vs mutant based on phenotypes my mutants actually had a smaller band and upon sequencing I found something like a 4 bp deletion. The hets still had the larger band, and I never saw something homozygous for the large band. I tried TOPO cloning and PCRing the het DNA but nothing had an insertion.
So long story short I would go ahead and sequence those 800bp bands and see what you get.
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