Dear fellows,

i need help regrading plasmid curing of my yeast. i am working with klyveromyces sp. my research is cloning and expression of killer toxin protein by this strain. i wana confirm ethir the gene of particular killer protein is on the yeast palsmid or chromosomal one. becuase it has already been reported that some spp.of klyveromyces produce killer toxin protein and the gene os on plasmid.

i wana cure my plasmid by ethidium bromid.i have to use different concetration of EB and keep my culture shaking for one day and the CFU and at last i have to confir plasmid absence by selecting random colonies.

my question s that how to make EB solution and at what stage it should be added?

i mean first i have to make YPD cultre EB and then introduce my yeast strain or

first i have to culture over night then add this already culture solution in new YPD with EB and keep it shaking for over night or what?

i am reallly confused about this please help me this with detail protocol

i shoul be very thank full to you all

regards

Aslam