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From: werner@aecom.YU.EDU (Craig Werner)
Newsgroups: sci.bio
Subject: Hints on Plasmid preps
Message-ID: <1172@aecom.YU.EDU>
Date: Fri, 3-Jul-87 02:46:18 EDT
Article-I.D.: aecom.1172
Posted: Fri Jul  3 02:46:18 1987
Date-Received: Sat, 4-Jul-87 19:47:41 EDT
References: <1137@aecom.YU.EDU> <404@uhnix2.UUCP> <1161@aecom.YU.EDU> <1485@sigi.Colorado.EDU>
Organization: Albert Einstein Coll. of Med., NY
Lines: 31


	The following hint came about after I was consistently able to
get 10 times as much plasmid (pUC18) DNA from a 1.5 ml (Eppendorf tube)
miniprep than any one else on our floor. I was averaging about 20
micrograms per tube - 1/500 of the prep was visible after after
digestion, whereas others were digesting half or all of the prep to
see bands.
	What did I do differently?

	Everybody else used old cultures or cultures put on a roller,
or just in the 37C incubator overnight.
	I started instead doing the following:  

	Placing 5 ml of NZYM into a 250 ml Erlemeyer flask and inoculating
that, letting it shake vigorously overnight, and doing the plasmid prep
first thing in the morning.  I credit it all to aeration, although like
everybody else in biology, I changed a few steps later in the protocol
as well.


	Also,  concerning gels:  normally 1 mg/ml Ethidium Bromide is
used as 2000X solution (i.e. 50 ml gels get 25 ul EthBr).  I've been
using 12 ul for years (4000X) with no adverse results, and I just went
down to 8 ul (6000X) ditto.  L prefer it because you don't see the 
background haze at the top of the gel.  The fact that it also throws
less carcinogens around lab is also a positive point.
-- 
	   Craig "Baby Doc" Werner   (future MD/PhD, 3 years down, 4 to go)
	     werner@aecom.YU.EDU -- Albert Einstein College of Medicine
              (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517)
               "Time flies when you're streaking out N. gonorrheae."