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From: werner@aecom.YU.EDU (Craig Werner)
Newsgroups: sci.bio
Subject: Re: Trick on Hanahan transformation
Message-ID: <1118@aecom.YU.EDU>
Date: Sat, 6-Jun-87 22:37:52 EDT
Article-I.D.: aecom.1118
Posted: Sat Jun  6 22:37:52 1987
Date-Received: Thu, 11-Jun-87 03:02:48 EDT
References: <9543@duke.cs.duke.edu> <1125@ius2.cs.cmu.edu> <701@edge.UUCP> <2207@husc6.UUCP>
Organization: Albert Einstein Coll. of Med., NY
Lines: 17


	I never thought the following would make a difference, but in fact
it makes a 20-fold improvement.
	The SOC medium (SOB + glucose) that is added to allow the cells
to recover and express resistance is very important, or the pH thereof
is.  If you just mix it, the pH is about 7.2.  One time I pH'd it to
7.4 ala Maniatis and the transformation frequency plumetted. So I looked
up the paper. Hanahan's SOC was kept between pH 6.9 and 7.0.  I then
did the same.  The result: 20-fold enhancement over pH 7.2, about 
100-fold over the disastrous 7.4.  
	It just goes to show.  Even if the cells take up DNA, if they
proceed to die, you get poor results.
-- 
			      Craig Werner (MD/PhD '91)
				!philabs!aecom!werner
              (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517)
                      "If I don't see you soon, I'll see you later."e