plasmid isolation from yeast
boutell at cf.ac.uk
Fri Sep 13 10:06:40 EST 1996
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
>> 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 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
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.
Joe Boutell. (no affiliation to QIAGEN, but a free t-shirt or keg of beer
wouldn't go amiss!!?).
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