I want to grow yeast (BY4743) with a certain knock out (deletion), the deletion cassette is KANMX4. I assume I have to use 200mcg/ml geneticin.
But can anyone explain step by step how i have to proceed with the ordered strain - it comes on agar in little vials.
Do the agar plates I am streaking them onto need to contain geneticin?
If I culture them in liquid do I always have to add geneticin?
Cause I read here sometimes that people use genetecin only on the replica plates.
Can someone just tell me how I need to proceed with the cells, so the protein of interest is really deleted/knocked out and the KANMX4 deletion cassette is working.
Thank you
Because this is a stable integration in the chromosome, you don't need to maintain selective pressure like you would if it was a plasmid. So you really do not need to use geneticin to grow the strain. I would just streak it out on a normal rich plate (or appropriately supplemented minimal plate if there are auxotrophic markers) and then I would test a few colonies on geneticin plate to make sure all the colonies carry that marker. I would also double check for the knockout, either the phenotype if there is a good one, or by PCR to be sure the strain carries the proper knockout.
One you have a stock from a single colony where the markers are confirmed, it should be fine to use without selection.
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