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From: lonetto@phri.UUCP (Michael Lonetto)
Newsgroups: sci.bio
Subject: Re: Hints on Plasmid preps
Message-ID: <2780@phri.UUCP>
Date: Fri, 10-Jul-87 17:50:02 EDT
Article-I.D.: phri.2780
Posted: Fri Jul 10 17:50:02 1987
Date-Received: Sun, 12-Jul-87 12:42:41 EDT
References: <1137@aecom.YU.EDU> <404@uhnix2.UUCP> <1161@aecom.YU.EDU> <1485@sigi.Colorado.EDU> <1172@aecom.YU.EDU>
Reply-To: lonetto@phri.UUCP (Michael Lonetto)
Organization: Public Health Research Inst. (NY, NY)
Lines: 17
Summary: for those doing LOTS of minipreps


Along the same lines as Craig's tips on mini preps:  we have often had
to screen >>100 transformant lines (my record was 400)for proper
restriction pattern, which puts a great deal of strain on shaker space
and glassware if you use flasks.  Our answer was to use "potatoe tubes",
25x175 mm glass culture tubes, with 1-2 ml of media, and shake
vigorously (300-350rpm) overnight.  One critical variable is the length
of incubation:  Too long and you're sunk (13-15 hrs worked well for 2 ml
cultures of JM105/pUC).  Another was the efficiency of lysis (make up
the lysozyme fresh).  Working this way I was able to determine restriction
maps using 0.5-1% of the yield from 1 ml of culture (one miniprep gives
plenty of material to purify a fragment and reclone it, a great timesaver).

-- 

Michael Lonetto    UUCP:(allegra!phri!lonetto) 
USMAIL: Public Health Research Institute, 455 1st Ave, NY, NY 10016