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Yeast Marker Swap Plasmids (pLU12 - Cross 1997 Yeast)

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#1 callanas



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Posted 09 August 2011 - 10:03 AM

Hope someone can help me with this!

I'm trying to swap the leu2 gene on a plasmid that I currently have in a yeast strain (leu/his/trp/ade/ura). I transformed the cells with a bam/sal digested pLU12 described in Cross 1997 and got about 50 transformants that were Ura+. Upon streaking these cells onto -ura/-leu, essentially all the colonies grew suggesting that the 'swap' occurred at the genomic level. I'm re-streaking the Leu+ colonies on to fresh -ura/-leu plates just to make sure they are in fact able to grow in the absence of leu. Has anyone experienced this low efficiency of episomal plasmid swapping (even Cross said it was pathetic, and I do not have those E. coli strains that select for plasmids.. sad...)

Also, I was reading a second marker swap strategy (Voth, 2003 Yeast) where they linearized both the recipient and donor swap plasmids. I do not have access to their plasmids but i was thinking I could do it with the pLU12?

Any advice would be stellar, thanks :)

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