plasmid isolation from yeast

[Joe Boutell]

kmorano at umich.edu (Kevin A. Morano) wrote:
>In article <klennon.410.32309D1C at acs.bu.edu>, klennon at acs.bu.edu wrote:
>
>
>> I am desperately seeking a protocol for isolating plasmid DNA from yeast, 
>> without having to isolate genomic DNA and then transform bacteria.  Can
>anyone 
>> refer me to information about doing so? I would be extremely appreciative!!
>
>Read the protocol in the Big Red Book (Current Protocols).  Basically, take
>1-2 ODs of cells, r/s in the cracking buffer and vortex with equivalent
>amount of phenol/chloroform.  See the Book for buffer recipe.  Spin and
>transform comp. e. coli with 2-5 ul of sup.  Voila.
>
>Kevin
>>
>
>-- 
>Kevin Morano, Thiele Lab
>University of Michigan Medical School
>kmorano at umich.edu


Yeah, but what was wanted was a protocol where you don't have to 
transform the stuff back into E.coli. In which case i would direct them 
to their local QIAGEN rep., who a while ago gave me an experimental 
protocol for isolating plasmid DNA from yeast (ie. they haven't optimised 
it fully):

1. Harvest 2 grams of yeast cells (wet weight) by centrifugation and 
rinse in water.

2. Resuspend cells in 4ml of freshly prepared SCE buffer (SCE=1M 
sorbitol, 0.1M NaAc, 60 mM EDTA, pH7.0)

3. Add 8mg zymolase enzyme and 30 microlitres of beta-mercaptoethanol, 
incubate 1 hour 37 C.

4. Spin down spheroblasts 5000rpm for 5 minutes.

5. Resuspend in 4 ml QIAGEN buffer P1, incubate 37 C for 15 min.

6. Add 2 ml lysis buffer (3% SDS, 60 mM EDTA). Mix gently but thoroughly, 
incubate 20 min 65 C. Volume should be 6 ml.

7. Add 3 ml of 3M KAc, pH5.5, mix immediately to avoid localized PDS 
precipitation, incubate on ice 15 min.

8. Centrifuge 4 C for 30 min at >30,000g. Remove supernatant promptly.

9. Follow QIAGEN Plasmid Midi protocol starting at equilibration step.

Cheers,
Joe Boutell. (no affiliation to QIAGEN, but a free t-shirt or keg of beer 
wouldn't go amiss!!?).