YipLac211 difficult integration in yeast (View forum version)



scaetano

Posted 29 May 2014 - 03:29 AM

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


CovanceABP

Posted 25 June 2014 - 08:28 AM

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...