Help - yeast plasmid rapid miniprep needed!
marcusj at helix.mgh.harvard.edu
Mon Apr 15 10:58:05 EST 1996
In article <stephan.witte-140496120458 at marvin.biologie.uni-konstanz.de>,
stephan.witte at uni-konstanz.de (Stephan Witte) wrote:
< After a two hybrid/interaction
trap screen I have to isolate plasmids
< from my positive clones.
< I didn«t succeed with a zymolase protocol, I suppose the enzyme does not
< lyse the cells.
< Are there other reliable protocols, maybe something like alkaline
< lysis...... (not much YEAST experience :-()
I've had great luck using the zymolyase method followed by Geneclean,
resuspending in a final volume of 12ul. Then I transform 80ul coli with
2ul DNA; you might want to try varying the amounts between 1ul and 5ul.
This is just using homemade bugs at around 10^6 to 10^7, not ultracomps --
and I always get around 50 colonies per plate. The only problem with this
method is that it is not rapid.
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