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A problem with deleting URA3 from S. cerevisiae


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#1 YBH

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Posted 28 March 2009 - 12:12 AM

Hi everyone,
I have been trying for the past 4 months to delete the gene URA3 from S. cerevisiae strain with no success. In order to delete the genes I use a PCR product from 2 strains with a deletion of URA3 that I transform into my cells. I have been trying different lengths of PCR products (200,300,500bp) and different ODs of transformation. I also have a positive control of cells that can do the deletion of URA3.
I use a 5FOA plates for selection of transformants.
Do you think that a prolonged incubation in YPD prior to seeding the cells on 5FOA can help the deletion?
Are there any other offerings for getting the job done?
Thanks.

#2 kajmak

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Posted 05 April 2009 - 01:58 AM

I have never that deletion, just plain plasmid transformation and glucose vs galactose selection. But, after each transformation I plate my yeast on glucose, incubate for 4-5 days, and then streak it out on galactose.

So, if you transform yeast with non-functional ura fragment, that yeast probably needs some time for gene exchange, therefore ypd media following transformation might help.

#3 YBH

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Posted 05 April 2009 - 04:32 AM

I have never that deletion, just plain plasmid transformation and glucose vs galactose selection. But, after each transformation I plate my yeast on glucose, incubate for 4-5 days, and then streak it out on galactose.

So, if you transform yeast with non-functional ura fragment, that yeast probably needs some time for gene exchange, therefore ypd media following transformation might help.


Hi kajmak and thanks for your answer.

I recovered my cells for 4 hrs in liquid YPD prior to growth on selection media, but it might not be enough. Now I am trying to recover my transformed cells on YPD plate O/N, and following your advice I will prolong the time of recovery to 1,2,3,4,5 days. I hope it will work better.




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