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From: srp@ethz.UUCP
Newsgroups: sci.bio
Subject: Re: Life Classification ...further comments
Message-ID: <105@bernina.UUCP>
Date: Thu, 4-Jun-87 03:01:43 EDT
Article-I.D.: bernina.105
Posted: Thu Jun  4 03:01:43 1987
Date-Received: Sun, 7-Jun-87 01:55:00 EDT
References: <9543@duke.cs.duke.edu> <1125@ius2.cs.cmu.edu> <701@edge.UUCP> <1211@sigi.Colorado.EDU> <1105@aecom.YU.EDU>
Reply-To: srp@bernina.UUCP (Scott Presnell)
Organization: Chem. Dept., Swiss Federal Inst. of Tech. (ETH-Zurich)
Lines: 35

In article <1105@aecom.YU.EDU> werner@aecom.YU.EDU (Craig Werner) writes:
>	Escherichia coli, the darling of molecular biologists does
>not undergo natural transformation.  It was known for many years that
>DNA could be introduced by spheroplasting cells, but regeneration
>of bacteria was inefficient.
>	It was only in 1973 that a method (the CaCl2 at 0C technique)
>was demonstrated that allows high efficiency DNA transfer into E. coli.
>This is far from natural, and less than half of the cells remain
>viable after treatment. Still with 10^10 cells/ml of transforming
>mixture, factors of 2 aren't really significant.  Douglas Hanahan,
>more recently, has described methods using Ca, Mn, Cobalt Hexamine
>Chloride, DMSO, and DTT, and other reagants that I can't recall right
>off, that boost transformation frequencies several orders of magnitude -
>to almost a percent or higher.

You got most of the reagents, however even though there is a table
reporting accross the board success with 'normal' strains in the paper, in
my hands (and hands of other lab members), the procedure works "several
orders of magnitude" better only with his own strains, the DH series
(which, BTW, are _rumored_ to spontaneously mutate/revert or be otherwise
unstable, even though it is RecA-!!). 

The technique does have the advantage of being a one-day procedure,
whereas there is a 24 Hr. wait for the CaCl2 procedure (for competency to
set in).

It should be pointed out also that transformation efficency is also
related to size of DNA being transformed, in a logarithmic relationship.

Regards,

Scott Presnell	(Scientific Mercenaries Inc.)		Organic Chemistry
Swiss Federal Institute of Technology  (ETH-Zentrum)
CH-8092 Zurich, Switzerland.
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