prep of DNA from Biolabs' vent-pol 10x buffer?

Michael Szardenings msz at bio.embnet.se
Mon Jan 22 05:19:17 EST 1996


Dear netters,
we very recently ran into trouble with the preparation of PCR-fragments
for the mega-primer mutagenesis approach.
We routinely use a kit from Genomed, called Jetpure, to prepare the PCR-
fragments and to remove oligonucleotides at the same time. This has been
working extraordinarily well (and quick), although Genomed claims, that
yields with buffers containing detergents may be low. The latest batch
Vent-Pol&buffers opened by us indeed resulted in such problems, after
about 1-2 years successful work with Vent in combination with the kit. The
yields are now about 10-20% regardless which size the PCR product is.
We believe, that something in the Biolabs buffers must have changed. I
tried to get advice from them by e-mail, but they say, it can't (?) be
true. On the other hand, what should have gone wrong?
The formulation of the 'Jetpure' kit from Genomed has definitely not changed.
We have used the same kit on and on, when suddenly these problems occured. I
have been talking with the people from Genomed. They told me, that these
problems can't be due to the kit either. Which is actually rational, as they
did not change the recipe and we even observed the loss of efficiency with the
same kit being in use. There is no way, that the kit may go bad (it were
actually 2 kits in use at the same time!), the reagents ought to be stable for
decades. I can not believe that Biolabs did not change anything, as the only
thing we changed in the last 2 months was opening a new charge of Vent-Pol.
Has anybody out there made similiar experiences in the last months?

Regards
                Michael

================================================================================
*       *   ******   ********  *************************************************
**     **  ***   **  ********  * Michael Szardenings                           *
***   ***  *****         ***   * Biomedical Center, Pharm. Pharm.              *
**** ****     *****   ***      * Box 591                Tel.:int46-18-174224   *
** *** **  **   ***  ********  * S-751 24 Uppsala       FAX :int46-18-559718   *
**  *  **   ******   ********  *************************************************
================================================================================
INTERNET : MSZ at bmc.uu.se
================================================================================

Disclaimer 1: I have no connections to any of the companies mentioned
above, nor do I make any profits by telling that their products may be
sometimes not the best.... :-)

Disclaimer 2: Don't b(f)lame me for using kits, I purified the DNA on a
gel before and the quality was just worse, yields (were) lower and time...



More information about the Methods mailing list