Hi,
I am trying to use px458 plasmid (Cas9-2A-GFP) to generate crispr KO cells and wonder if there's anyone having the experience could suggest the starting number of cells for screening. If the GFP positive cells are about 30%, usually how many cells will be used for FACS sorting and how many 96 well plates will be generated after sorting into them? If we want to increase KO efficiency, will it help if the top 10-20% GFP cells are selectively collected? Thanks for your time!
It really depends on how well the cells grow in isolation. I would use anywhere from a 6cm to a 10cm confluent plate and generate about 2-4 96-well plates. I would seed in no more than 100ul media per well.
Hi Catalina,
Thanks for answering. I will try the scale you suggest.
Best,
Wen An
- Badges
- Science topic