From owner-diagnostics@net.bio.net Tue May 06 23:00:00 1997 Path: biosci!biosci!not-for-mail From: Greg Forbes Newsgroups: bionet.diagnostics Subject: PAGE problem Date: 7 May 1997 01:58:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Approved: odonnell@sasa.gov.uk Distribution: world Message-ID: <5kpg8i$37c@net.bio.net> NNTP-Posting-Host: net.bio.net GPI is a useful marker for plant pathogenic fungus Phytophthora infestans. Some 7 alleles have been identified, all of which are known by their migration distances relative to a standard allele which was given an arbitrary value of 100. Most alleles can be easily identified on cellulose acetate and Tris-Glycine buffer, and/or on starch gels with TC6 tank buffer and His 6 gel buffer. Unfortunately, the 90 allele can not be easily distinguished from the 100 allele using these systems. Following advice from a collegue, we've been using 12 or 14 % acrylamide gels, with Tris-Borate pH 9.0, Tris-Glycine pH 8.5 or 8.8 as tank buffers, and Tris-HCl pH 8.9 as gel buffer, with or without a stacking gel, Tris-HCl pH 6.9. All this has been done on 1mm thick gels in a vertical system. The enzyme migrates really slowly, only about 2cm when the bromophenol blue has run off the end of the gel (16cm) and the only alleles to separate well are 100/122, which separate much better in starch. There`s no streaking and the bands are quite neat, we I don`t think there`s anything wrong with the extracts. We're about to try reducing the % of acrylamide, but if anyone can help resolve this problem with some other usaful suggestions, we'd be very grateful. Greg Forbes Lynn Erselius Greg Forbes International Potato Center (CIP) P.O. 17-21-1977, Quito, Ecuador Tel. +593-2-690362; Fax +593-2-692604 .