FST
production
A
protocol for FST production based on "gene-walking" (Devic
et al.. 1997), was optimized for a large scale of an amplification
and a systematic sequencing (Balzergue S, Dubreucq B et al.. 2001).
The DNA fragment(s) resulting from the PCR were directly sequenced
from the left or the right border to determine the genomic sequence
flanking the insertion. T-DNA derived sequences were removed.
All these genomic data are managed in the data base FLAGdb++ (link
http://193.51.165.9/projects/FLAGdb++/HTML/index.shtml) developed
by the Bioinformatique team of INRA-URGV (Samson F et al.. Nucleic
AcidsRes. 2002 Jan). Produced FSTs, as well as the corresponding lines,
are accessible for the international scientific community via the
FLAGdb++ Web site.
To date, a total of 41313 FST were generated and a deposit of approximately
30 000 FST in the EMBL web site was carried out. A study on the first
9000 FSTs brought informations on the mechanism of integration of
the T-DNA in the A. thaliana genome (Brunaud V et al.. 2002).
The requests for T-DNA lines corresponding to your favourite FST is
possible through the pages "Ask for line" of the FLAGdb++
Web sites or through the publiclines interface (link http://dbsgap.versailles.inra.fr/publiclines/)
To
confirm your T-DNA insertion
-Note
if the FST was amplified with the left border (LB) or the right border
(RB) of the T-DNA.
-Amplify
by PCR (standard program) on the diluted DNA (1/50 in general), a
flanking fragment of the insertion. Use one specific oligo for genomic
sequence. (Since rearrangements during the translocation event can
occur, if possible, design a primer directly from the FST sequence)
and one primer from the T-DNA border :
For
the RB, you can use
RB4
5'-TCACGGGTTGGGGTTTCTACAGGAC-3'
or
Tag3 : 5'-CTGATACCAGACGTTGCCCGCATAA-3'
For
the LB, you can use:
LB4
5'-CGTGTGCCAGGTGCCCACGGAATAGT-3'
or
Tag5 :5'-CTACAAATTGCCTTTTCTTATCGAC-3'
Confirm
your insertion by sequencing the purified PCR product.
