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How to determine the number of T-DNA insertions at a locus

LIN BAIQING lbaiqing at toulouse.inra.fr
Wed Oct 15 11:27:39 EST 1997

Hi, Gilles,
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

Baiqing Lin
INRA, Toulouse

> 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
> Thanks
> Gilles Vachon
> Laboratoire de Genetique Moleculaire des Plantes,
> UMR 5755, CERMO
> Universite J. Fourier, BP 53X
> Tel: (33) 76 63 56 58
> fax: (33) 76 51 43 36
> e-mail: Gilles.Vachon at ujf-grenoble.fr

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