We are screening T-DNA library from Versailles. I always do Southern
blotting to determine if the our candidate mutants containing only one
functional insertion. I synthesized two probes from T-DNA which spanned
the left and right borders respectively. Using an enzyme to cut T-DNA
into two segments, do two hybrids. If you got only one positive signal,
it seems the 3:1 KanR trait be due to only one functional insertion.
Hope it will be helpful
> Hi all,
>> We are facing the problem of determining the number of T-DNA insertions at
> a locus. Our lines carrie a GUS gene under control of a specific promoter
> and segregate 3:1 regarding Kanamycine resistance.
> What are is the best strategy for doing so and avoid problems in
> interpreting the results (tandem insertions etc..)? cut inside GUS? with
> one enzyme, two enzymes.
> Help or references are appreciated
>> Gilles Vachon
> Laboratoire de Genetique Moleculaire des Plantes,
> UMR 5755, CERMO
> Universite J. Fourier, BP 53X
> 38041 GRENOBLE CEDEX
> Tel: (33) 76 63 56 58
> fax: (33) 76 51 43 36
> e-mail: Gilles.Vachon at ujf-grenoble.fr>