URA3 pop out help, thanks! - (Mar/29/2009 )

Dear All,

I am currently struggling with the URA3 gene. I thought my strain didn't have the URA3 gene. Actually, it has it since I got URA3 PCR product. I realized that the strain my boss got from other labs has been inserted a FLAG tag at C- terminal of one gene using the plasmid YIplac211 which has a URA3 gene.

I don't know how they constructed the integrating plasmid, but I think there are two possibilities. One is that they PCRed both C-terminal portion and the stop coden downstream portion of the gene separately and then added FLAG tag between the C-terminus and downstream region. In this case, the URA3 can be popped out by 5FOA selection leaving the FLAG tag in the genome.

The other possibility is that they PCRed the C terminus and then insert into YIplac211 which contained sequences encoding the FLAG tag followed by the terminator sequence of the CYC1 gene. My question is if this is the case, can I looped out the URA3 by 5 FOA selection but leaving the FLAG in the genome? I still need the FLAG tag for my following experiments.

Thanks a million for your help in advance!

Cy

Dear all,

Please help me! probably this is a stupid question. but as a beginner for yeast genetics, I am not sure the answer. I think that in the second case, the FLAG tag won't leave in the genome after 5 FOA selection, since the repeat sequences (3' portion of the gene) are flanking FLAG and URA3. In my mind, if there is repeat sequence flanking only URA3, then the URA3 can be looped out leaving the tag in the genome. I was wondering if this is true.

Thanks for your comments in advance!

cy