PAGE problem

Greg Forbes forbes at cip.org.ec
Wed May 7 03:58:58 EST 1997


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



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