We are interested in generating knockout in cell lines using the CRISPER/CAS9 system and replacing our gene with GFP. After performing a double transfection using the guide RNA and the donor vector, we selected the cells against purmycin and selected a number of colonies. How would you go about confirming the knockout?
Thanks Han. We tried that, but can you be sure if its mono or bi allelic insertion with just immunoblotting?
Hi Mohamed, you can estimate the status of your clones by immunoblotting. For example, if mono-, I mean ideally, you should see the weaker band of your target gene; if not, you should see nothing. However, this way is just for your estimation. If 90% of cells are bi-allelic and 10% cells are mono-, you may also see nothing when doing immunoblots.
Yes I agree with sequencing will solve this issue. we designed primers from the genomic region spanning the GFP and that confirmed the insertion.
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