From owner-celegans@net.bio.net Wed Oct 29 22:00:00 1997 Path: biosci!biosci!not-for-mail From: Leon Avery Newsgroups: bionet.celegans Subject: Re: making males Date: 30 Oct 1997 06:15:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Approved: wmorgan@acs.wooster.edu Distribution: world Message-ID: <63a4pp$kun@net.bio.net> NNTP-Posting-Host: net.bio.net Heat shock is a very touchy protocol. You have to get the time exactly right: half an hour too long, and the worms are sterile, half an hour too short, and there's no nondisjunction. Furthermore, the correct time depends very sensitively on the temperature, and you seldom know the temperature accurately enough. So I do as follows: put 5 L4 hermaphrodites on each of 6 plates at 30C. Take out two plates after 5.5 hours, two more after 6 hours, and two more after 6.5 hours. One of those pairs should work. You'll have advance warning which, because you'll usually see some vulval abnormalities in the parents, and they will produce small broods. If all your plates are sterile or if all are normal, adjust your incubator accordingly. The frequency of males is still not high -- you have to hunt for them. -- Leon Avery (214) 648-4931 (voice) Department of Molecular Biology and Oncology -1488 (fax) University of Texas Southwestern Medical Center 5323 Harry Hines Blvd leon@eatworms.swmed.edu Dallas, TX 75235-9148 http://eatworms.swmed.edu/~leon/ .