From owner-drosophila@net.bio.net Tue Mar 07 22:00:00 1995 Path: biosci!LAMAR.COLOSTATE.EDU!ashok From: ashok@LAMAR.COLOSTATE.EDU (Ashok Mudgapalli) Newsgroups: bionet.drosophila Subject: HELP PLEASE!! Date: 7 Mar 1995 19:11:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear netters I am doing drosophila embryo transformations using a cDNA clone. I am getting poor or no survival of Ist instars when I did not even injected with injection buffer. The exact method which I follow is....I discard Ist 1 hr collection of embryos and collect next batch of embryos for 20-30 min. I dechorinate them in 50% chlorax (treatment for 70-80 sec) and wash in deionised water for at least 8-10 time, just to make sure that there is no trace of chlorax on the embryos. Then I give a gentle wash with sterile DD water. I place the healthy embryos on double stick tape using a fine brush. Double stick tape is from "Scotch" Cat.136. After placing the embryos on tape I dessicate for 11 min (which is appropriate for the existing humidity conditions). After dessication I place halocarbon oil (series 700) on the embryos and am leaving the slide in moist plastic box at RT (22 C). Even after 48-60 h I do not see at least 10% hatching. When I looked them under microscope time to time during embryogenesis, they were perfectly completing the development in about 48 h and you can see the developed larvae moving in the vitelline membrane. After this step none of them are emerging and they are dying.I am also checking the oil on the embryos from time to time. One thing I noticed about tape in the oil is, it is turning in to thick milky white color after 48 h (i.e., almost at the time of completion of embryogenesis). I do not know where the problem is lying. Is it the brand of doulbe stick tape I am using responsible or some thing else? As I mentioned these embryos were not even injected. I greatly appreciate suggestions and comments from netters. If any body has recently used particular brand of double stick tape which worked well for them, I appreciate if they can send me a small portion of the tape as a gift. Thanks in advance Dr.Ashok Mudgapalli Dept of Biology Colorado State University Fort Collins, CO 80521 .