From owner-celegans@net.bio.net Mon Mar 30 23:00:00 1998 Path: biosci!biosci!not-for-mail From: chaser Newsgroups: bionet.celegans Subject: Rescue of Lethal Mutation by Cosmid Injection Date: 31 Mar 1998 07:06:47 -0800 Organization: K-JIST Lines: 33 Sender: daemon@net.bio.net Approved: wmorgan@acs.wooster.edu Distribution: world Message-ID: <6fr0q7$fg5@net.bio.net> NNTP-Posting-Host: net.bio.net I am a student in K-JIST in Korea. We've got several embryonic lethal mutations near the Unc-29 region on LG I. [ unc-29(e193) Let/ dpy-5(e61) unc-13(e1091) ] I. Currently the possible mutation region was narrowed down around 15 cosmids. Now, I'd like to find out a cosmid containing lethal mutation by germ-line transformation. I am familliar with microinjection technique however, I am really worrying ahout this rescue experiment because of my short knowledge and experiance. Therefore I would appreciate if you could give your great information to me. If you are willing to help young student, please answer fallowing questions. 1. There are several overlapping cosmids containing same sequence. Is there any criteria in selecting cosmids? 2. Could I get a protocol for cosmid preparation? Do cosmids need special purification procedure for injection? 3. What quantity ratio would be suitable between cosmids and marker plasmid(rol-6) in injection mixture? 4. What kinds of control experiment are possible? (i.e. rol-6 only injection) 5. On which generation should I observe the phenotypic rescue after cosmid injection? 6. In our mutant strains, recombination is so frequent that the lethal mutation is often disappear. Therefore, how can I distinguish rescue of lethality from this recombination after cosmid injection? 7. Could you recommend some references which can rescue me from obscurities? .