Yeast plasmid preps

I am working with a gene which is very toxic to E. coli, so I would like to
do as much of the work as possible in yeast. In particular, I would like
to do the constructions with yeast gap repair cloning and then prepare the
plasmid DNA in yeast. Because I want to use this DNA for transfection
experiments, I need a fair bit of it - on the order of 10 ug would be a
minimum. It seems that the vast majority of the yeast plasmid preps are
geared for those who just need enough DNA to transform E. coli and thus are
inappropriate for my uses. However, I have seen several commercial kits
that promise high enough yields to make this feasible. For example Omega
Biotek's EZNA prep promises 1 ug of plasmid from a 5 ml yeast culture and
CPG's DNA-Pure Gram+/Yeast Plasmid Mini-Prep Kit promises 2-3 ug from 10
ml. We have tried the EZNA kit several times and not observed anywhere
near the expected yields.

We have also tried a prep in which we use lyticase to produce spheroplasts
and then lyse them and use a Qiagen miniprep to purify plasmid. This gave
us only about 20 ng from a 5 ml culture. I suspect that our spheroplast
production may be a limitation in the Qiagen prep, since when we tested
them in water, we only saw about 1% of them lyse. Our cell wall digestion
conditions were: 5 ml of cells spun down and resuspended in 50 ul of 2
mg/ml lyticase, 1 M sorbitol, 0.1 M NaOAc, 60 uM EDTA at 37 C for 1 hour.

So my question is whether anyone has had success with any of these plasmid
preps (or any others) in making large quantities of plasmid DNA in yeast.
If so, I'd much appreciate protocols or suggestions on how to do this. In
particular, are there better ways to prepare spheroplasts?

Thanks,
Tim Caspar



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