2 replies to this topic

[YBH]

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.

[kajmak]

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.

[YBH]

kajmak, on Apr 5 2009, 02:58 AM, said:

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.