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? I'm new to the list, but in searching the archives, it
seems the last time anyone raised an issue like this was in 1997 and
the answer referenced a 1987 paper in which the plasmid was prepared
by making spheroplasts and then using a standard (for that time)
alkaline lysis plasmid prep utilizing a CsCl gradient. I'm hoping
there's something new and better since then.

Thanks,
Tim


______________O_________oO_____________oO______o__ _____oO___________________
YEAST bionet newsgroup see: [Only registered users see links. ]
YEAST e-mail: messages sent to [Only registered users see links. ]
subscribe: mailto:[Only registered users see links. ].net and say: subscribe yeast
unsubscribe: mailto:[Only registered users see links. ].net and say: unsubscribe yeast
YEAST on the WWW: [Only registered users see links. ]
Newsgroup Moderator SGD Curators: [Only registered users see links. ]