Hello,
should one validate and backcross knockout strains from CGC that originated from EMS mutagenesis? Or do you use it as is? If validation + backcrossing are necessary, do you know where I could find a protocol for that?
Thanks a lot!
Each backcross (only) removes half the genetic background., and thus it is good to backcross a few time but remember you will never be sure. In the old days one wanted to have rescue with a transgene to confirm the causal mutation. I think now it is easier to just make another knockout with CRISPR. Also, in a few cases, we have made isogenic controls by reverting a STOP-IN insertion back to the wild-type sequence.