Can anybody please explain how to do the selection step? I am currently growing my cells in 6 well plate and after puromycin treatment, I can see physical colonies (like the ones you see when you plate bacteria on agar) and no sign of contamination. do I pick the colonies and transfer to a T25 to grow them normally, or do I trysinize (detach) all the colonies together and dilute them in order to plate 1-2 cells per well of a 6-well plate?
I advise allowing the colonies to expand to the point that they are large enough to scratch passage. You then treat the cell media with ROKi for a few hours and the scratch passaging a selection of the colonies to 24 well plates for further expansion. Ideally the colonies will be large enough when you scratch passage to also collect some cell material for genomic DNA analysis and you will be able to immediately ascertain whether your CRISPR has worked. I usually try and passage 100-200 colonies and as long as the CRISPR cutting is efficient i will get plenty of hits.
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