Cloning in pGEM-T

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Thu Nov 25 11:53:59 EST 1999


Christophe Jacob wrote:
> 
> Does anyone know if the purification of PCR fragments (from agarose gel or
> with a column to eliminate other bands and/or oligonucleotides) before
> cloning in T/A vectors is a critical step ??
> 
> Thanks for yours answers.
> Regards,
> 
> Christophe.
> 
> --
> Christophe JACOB
> Lab. Forest Biology
> University Nancy 1
> BP 239
> 54506 VANDOEUVRE
> email: jacob at scbiol.u-nancy.fr

In my hands any purification step, especially gel purification, 
decreases ligation efficiency when doin T/A cloning. So, if you
have a single band you're lucky, if not than you might have to
do a lot of minipreps. 
The manual fo invitrogen's T/A cloning kit suggests to do the ligation
step within 24 h after you ran the PCR otherwise the A's tend to fall
of. Don't know whether this is true but it would explain things.

Ricky
*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de




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