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Yeast integration with leu2d1 and leu2 alleles


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#1 amano

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Posted 26 March 2009 - 02:02 PM

I am trying to find out more information about the leu2d1 allele. Specifically, I have a gene cassette in the prs305 Leu2 based integrating plasmid, and a yeast strain that has a leu2d1 allele. Is it possible to integrate with a single cut in the Leu2 portion of the plasmid? Is there enough homology between Leu2 and Leu2d1 to integrate and select with Leucine drop out media?

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#2 amano

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Posted 26 March 2009 - 02:49 PM

I was able to answer my own question, actually answered in Sikorski and Hieter Genetics 122: 19-27, 1989, which describes the construction of the pRS30X and pRS31X series.

Leu2d1 retains enough homology to allow for homologous recombination at the Leu2 locus. Interestingly so do ura3-52, trp1-d63, but NOT his3-d200, trp1-d1.

But now a followup question that I can't figure out -- multiple sources describe using the the same integration locus and selection marker. For example, digest prs305 with BstEII, which is in the Leu2 marker, for integration, but also select with Leu drop out media. Why is this?? Wouldn't integration at Leu2 disrupt the Leu2 locus, as well as the leu2 marker on the original integration plasmid? How does one get a functional Leu2 marker for Leu- selection?




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