Relay-Version: version B 2.10 5/3/83; site utzoo.UUCP
Path: utzoo!mnetor!seismo!gatech!hao!boulder!pell
From: pell@boulder.Colorado.EDU (Anthony Pelletier)
Newsgroups: sci.bio
Subject: Re: Life Classification ...further comments
Message-ID: <1302@sigi.Colorado.EDU>
Date: Fri, 5-Jun-87 14:13:49 EDT
Article-I.D.: sigi.1302
Posted: Fri Jun  5 14:13:49 1987
Date-Received: Tue, 9-Jun-87 07:29:37 EDT
References: <9543@duke.cs.duke.edu> <1125@ius2.cs.cmu.edu> <701@edge.UUCP> <1211@sigi.Colorado.EDU> <1105@aecom.YU.EDU> <105@bernina.UUCP>
Sender: news@sigi.Colorado.EDU
Reply-To: pell@boulder.Colorado.EDU (Anthony Pelletier)
Organization: University of Colorado, Boulder
Lines: 44

(Scott Presnell) writes:
re: hanahan protocol
>
>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-!!). 
>
Even Doug admits that the protocol works that much better only on the
DH cell lines.  For reasons that have never been clear to me, he does get
the protocol to work on many other cell lines better than I (or anyone I know)
have been able.  Something in his body temperature or "karma" I guess.
It is well established that HB101 will not stand up to the Hanahan proceedure.
It even says so in the "Joy of Cloning."
Concerning the _rumor_:  I use DH1s mostly.  I have had little trouble with
that.  But then I really don't care about the cell line so long as it
transforms well and doesn't screw up my clones.  I do routinely re-purify
the strain before preparing a large batch of competent cells.


>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).
>
Two points: In HB101 and others I have used with the CaCl2 proceedure,
the cells are competent immedeatly after treatment.  I usually get
5-10 fold increase in competence if I let them sit for a day.  I suppose
it depends upon how many colonies you need as to whether the extra log
is worth it.
Second, competent cells can be stored frozen for months.  
I'd be glad to send a protocol if you like; but it is fairly intuitive.
Just freeze in pre-cooled tubes in aliquots of .5-1.0 mls right
in the CaCl2.  Thaw on ice and use soon after thawing.
We keep competent stocks of several cell lines and have had no trouble
with any.
It is possible that your work requires greater efficiency than mine,
but if not, these changes will save you a fair bit of time.
happy cloning.

A.J.P. (Few degrees are worth remembering--and none are worth predicting)
is is is