advice needed: my PCR fragment gives low yield when grown up! Why?

R. John Lye rjl6n at virginia.edu
Mon Oct 29 07:59:55 EST 2001


Trond Erik Vee Aune wrote:

> You can try with another vector, but I would then try one with a
> different oriV than PBluescript just to test out if there's some
> reaction between your insert and replication initiation.

FWIW, I recently had a similar problem.  No matter how hard I tried
to subclone a piece into pBluescript, it just wouldn't go.  I finally switched
to Invitrogen's pZero-2 vector which uses a double selection scheme
(antibiotic resistance and disruption of a toxic gene) to increase the
chances of getting your clone.  This is what finally worked for me.
Maybe just switching ori's would have been enough, but this worked
so I didn't pursue the issue any further.

Hope that helps,
John




More information about the Methods mailing list