Frozen Electrocompetent Yeast, anyone?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Jul 25 21:11:06 EST 2001


hroychow at nmsu.edu wrote:
>Does anybody have any protocol that allows S. cerevisiae to be frozen 
>after the cells have been rendered electrocompetent? ... You know, like 
>the E.coli cells. I have not been able to locate any protocol that allows 
>for that luxury.

For Pichia pastoris, I've used essentially the same washing protocol as for 
E. coli with the exception that the final wash and resuspension was done in
1.0 M sorbitol. Invitrogen says you can freeze but efficiency drops 
dramatically. I also tried modification that purports to increase efficiency 
dramatically (before wash, treat with 100 mM DTT at pH 8.0). I compared
cells +/- DTT, +/- freezing. Result: DTT treatment is a lot more trouble
and expense yet only 20% better (but voltage was not re-optimized!). 
Freezing of cells not treated with DTT resulted in ~ less than 2X drop of
efficiency (more than acceptable!). Frozen DTT-treated cells did not
produce any transformants - I suspect they all died from zap. 

IMHO, there is absolutely no reason why freezing would not work
for Saccharomyces. I bet it will (oops! - did you try it? :-))

>I am not sure how to get rid of this "worm" and would 
>appreciate any help.

First and foremost, get good antivirus. F-Secure is the best, Norton 
and McAfee are the worst. 

Or, do a search for something like "trojan removal". There are a number
of such programs. It will cure youe PC. Or, if you like investigative 
passtime, you can hunt it down and kill yourself. There are freeware 
programs called "File Monitor" and "Registry Monitor". Using them 
should help you find out what file does these malicious things. 

HTH, 

        - Dima




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