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Abo-Ogiala, Atef
Jun 16, 2009, 7:34:45 AM6/16/09
to arab...@magpie.bio.indiana.edu
Please, I need to know how I can check the orientation of Arab. mutants using
web sites before PCR reaction. I mean to short the time and to avoid to do
the three primers compinations (Lp+Rp), (Rp+LBa), (Lp+LBa). I mean how I can
know that the T-DNA insertion inverted or not before the PCR reactions.
web sites before PCR reaction. I mean to short the time and to avoid to do
the three primers compinations (Lp+Rp), (Rp+LBa), (Lp+LBa). I mean how I can
know that the T-DNA insertion inverted or not before the PCR reactions.
Best regards
Atef Abo-Ogiala
Abo-Ogiala, Atef
Jun 16, 2009, 7:35:32 AM6/16/09
to arab...@magpie.bio.indiana.edu
I did the PCR reaction for one of Arabidopsis mutants to verify the T-DNA
insertion and the results are a bit strange. I mean I should get from the
first generation of the mutant at least one hetero plant but that is not
happened. The whole plants were wild type. So it could be there is no T-DNA
insertion or what? Another question concerning cDNA, I tested the cDNA with
the specific primers of my gene (Lp+Rp) and also with (Rp+LB) and I got
results in both reactions, however I got the RNA from wildtype plants. For
that I have a doubt that cDNA could be contaminated or I have no idea?
insertion and the results are a bit strange. I mean I should get from the
first generation of the mutant at least one hetero plant but that is not
happened. The whole plants were wild type. So it could be there is no T-DNA
insertion or what? Another question concerning cDNA, I tested the cDNA with
the specific primers of my gene (Lp+Rp) and also with (Rp+LB) and I got
results in both reactions, however I got the RNA from wildtype plants. For
that I have a doubt that cDNA could be contaminated or I have no idea?
Best regards
Atef Abo-Ogiala
Adrian
Jun 17, 2009, 4:22:04 PM6/17/09
to bionet-genom...@moderators.isc.org
On Jun 16, 7:35�am, "Abo-Ogiala, Atef" <Atef.Abo-Ogi...@forst.uni-
Use the SIGnAL T-DNA Express tool to find the T-DNA orientation for
your mutants. (http://signal.salk.edu/cgi-bin/tdnaexpress). As
recommendation avoid the use of LBb1 primer for Salk mutants, instead
use the LBb1.3 or LBa1 primer. Regarding to your PCR on cDNA just
repeat it but extract RNA from Arabidopsis WT plants grow up by
another person, if no possible grow a new batch of WT plants.
Hope this help!
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