Help, Yeast Technique-using knockout cassettes - transformations of yeast with knockout cassette (Jul/02/2005 )

Hi, I have done standard yeast transformations, but have never been successful thus far with using knockout cassettes. I have been cutting a plasmid containing a pho2::hisG::URA3::hisG cassette and tranforming it into yeast that are URA minus. thus far several attempts wit different concentrations are unsuccessful. Any thought? I have tried isolating and purifying the band and also just transforming the digest.
Any help would be much appreciated!

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Hi,
I think you are trying to knock out the hisG gene by homologous recomination! Do you? Then your problem is that Ura3 recombinates with the knocked out Ura3 gene in the genomic DNA because of the greater homologous region the cassette has with this area, than with hisG. So what you will have to do is creating another knockout cassette by using a resitence gene like zeocin or kanamycin instead of Ura3. Screening should be much more easier then, too, cause of the abilitiy of using antibotic selection.

i know other guys who had quite the same problems! so report about your success!

Greetings DerHenne

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