I am doing a KO RNP approach on suspension cell lines. I will be doing a limiting dilution to isolate single clones. My plan is to have 3 96-well plates plated with 0.5 cells/well, any comments?
I will then be doing a T7 endonuclease: I am having trouble getting the PCR product. From column purified genomic DNA, I am able to get the PCR product flanking the cut site. But direct PCR is not working.
My direct PCR protocol is a simple 1x10E3-1x10E5 cells resuspended in tris buffer and boiled for 10 mins, and using that for the PCR reaction.
Any robust and simple direct PCR protocol you recommend?