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[Automated-sequencing] pDONR vector

Alejandro Gonzalez agonzalez at gemabiotech.com
Tue May 9 10:22:05 EST 2006


Hi Ciaran, good morning. Yeah, we have same problems to sequence, but using
the pDONR201 vector because the same problem with the 2 identical homology
recombination sites. Invitrogen local support here informed us the problem
with this 2 large identical homology recombination sites, and they suggested
the pDONR221vector . We move to pDONR221 Invitrogen's vector, and we improve
the sequencing performance, it was Ok for us.
The problem with pDONR222 vector is with 2 recombination sites attB1 and
attB2 which share near identical homology ?

Thanks

Lic. Alejandro D González
Automatic Sequencing & R&D group.
Gema Biotech sa
M.T. de Alvear 2289
Bs. As., Argentina
+54-11-4825-8029 (137)


----- Original Message -----
From: "Fulton, Ciaran" <ciaran.Fulton at almac-diagnostics.com>
To: <autoseq at magpie.bio.indiana.edu>
Sent: Tuesday, May 09, 2006 4:50 AM
Subject: [Automated-sequencing] pDONR vector


> Hi all....we're using the pDONR222 vector for sequencing cDNA inserts from
libraries generated from the Invitrogen CloneMiner cDNA construction kit.
Generally we've had no problems with this set-up but we were talking to
another 3rd party recently who suggested that it might not be the most
suitable vector for sequencing. It has 2 recombination sites which share
near identical homology to one another and it has been suggested that if the
insert is less than 1kb then these can form secondary or hairpin structures,
essentially denying the primers template to prime off. There is supposed to
be a Biotechniques article about it somewhere but I haven't been able to
locate it.
>
> Has anyone any similar experiences using this or a derivative vector?
>
> Regards,
>
>
> Ciaran
>
> Dr. Ciaran Fulton
> Team Leader
> Sequencing R&D Group
>
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