Relay-Version: version B 2.10 5/3/83; site utzoo.UUCP Path: utzoo!mnetor!uunet!seismo!gatech!bloom-beacon!husc6!cmcl2!phri!lonetto 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