I have a CRISPR/Cas9 Wild Type (pSpCas9(BB)-2A-GFP (PX458)) plasmid that I have prepared, targeted for a nuclear protein's gene. I have already confirmed its sequence. I would like to test if it works before using FACS to isolate transfected clones. If I did a transfection on my cell line with this plasmid, would I likely be able to detect knock down of my protein of interest by Western Blotting, or will this be impossible without first isolating stable clones and confirming the mutation with SURVEYOR?
Thanks in advance!
Stephan, because the frequency of successful knockout is very very low, it is not obvious to detect knockdown of your target protein by WB in the cell pool. Now that you have confirmed your successful targeting by PCR sequencing, Isolation of stable clones should be the first and best choice for the following experiments. Once you get the stable clones, enlarge cultivation of them, then WB will get the desired results. Just for reference.
Stephan, because the frequency of successful knockout is very very low, it is not obvious to detect knockdown of your target protein by WB in the cell pool. Now that you have confirmed your successful targeting by PCR sequencing, Isolation of stable clones should be the first and best choice for the following experiments. Once you get the stable clones, enlarge cultivation of them, then WB will get the desired results. Just for reference.
You can run the surveyor assay on your pool of transfected cells before you start clonal isolation. This way you can get an estimate of what portion of the genomes, and by inference cells, in your population are mutated.
I like to knock out a larger chunk or use a targeted insertion. That makes it possible to see exactly what's happening with a simple PCR / gel electrophoresis step at the level of pool, heterozygous and homozygous clones.
Clontech's Guide-It mutation detection kit is superior to surveyor assay in my hand. It's way easy to do pcr and digestion than WB (also faster and cheaper). If your antibody is not great, your WB could be hard to interpret.
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