[Automated-sequencing] Re: pDONR vector

Phillip San Miguel pmiguel at purdue.edu
Tue May 9 12:52:58 EST 2006


Fulton, Ciaran wrote:
> 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

I don't recall ever using this vector. But I very much doubt that 
secondary structure is going to be much of an issue with current 
standard methods of sequencing.

pDONR222 has 31 base identical sequences, but they are in direct 
orientation with each other, not inverted. Also, wouldn't you use a 
recombination method to move fragments in? Unless I misunderstand the 
vector, seems like the recombination would remove these sequences along 
with ccdB and CmR.

Phillip SanMiguel
Purdue Genomics Core



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