Cloning in pGEM-T

Frederik Boernke boernke at
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
> email: jacob at

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.


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

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