YipLac211 difficult integration in yeast - (May/29/2014 )

Hello everyone,

I am trying to transform By4742 yeast strain with YipLac211 vector and it is not working. I know that this strain has the URA ORF removed, not with point mutations like other strains, and maybe it is hampering the integration process.

Until now I used the LiAc/PEG method (that works well with other transformations). 

Does anyone know a more efficient/specific transformation method for the efficient integration of this vector? 

Thank you in advance,

Scaetano

Hi Scaetano,

             I'm afraid that you will not be able to integrate the plasmid employing the URA3 locus in the strain (By4742) if it is carrying a full deletion of the URA ORF. Without the endogenous URA locus present in the genome, there is no possibility for crossing-over event to occur with the endonuclease digested vector you are transforming with (cut site within the URA3 locus). (Further, if you wanted to use the vector undigested as a CEN plasmid you need to have a mechanism on the plasmid like a CEN or 2 micron on the plasmid for the vector to be retained in the yeast strain). 

However, if the vector you are trying to integrate carries a yeast gene, then you could integrate using the locus of the gene itself. Be careful to select only a single restriction site within the yeast ORF and have the recombination even produce a full length ORF with the mutation/tag occuring downstream from the endonuclease site.

I hope this answers your question...there are troubleshooting approaches to the LiAc/PEG technique, but from your post, it sounds like you might need to redesign your experimental approach...