GATEWAY cloning of large PCR fragments

Oliver Berkowitz ollipolli at
Sun Sep 5 18:50:59 EST 2004

Hi Ricky,

how about cloning directly in a pENTRY vector. We have successfully cloned
2.3kb blunt-end (XmnI/EcoRV) into pENTR1A without any problems... Or if
reading frame allows it you can also create T-overhangs and try TA-cloning,
worked also fine for us...

Oli and Ricci

"Ricky Boernke" <ricky_boernke at> wrote in message
news:9652e74a.0409030554.3152522 at
> Dear friends,
> we're having a hard time creating an entry-clone for our favorite
> gene using Invitrogen's GATEWAY system. We've already tried two
> strategies. First, cloning of the PCR product into pENTR-D/TOPO, no
> colonies whatsoever. Second, PCR using attB primers and B/P into
> pDONR, no colonies. The PCR fragment is 3.1 kb which I feel is the
> cause for all the problems encountered. Does anybody here have experience
> with large fragments and GATEWAY? Is there anything special to
> consider in this case?
> Any input welcome.
> Have a nice weekend
> Ricky

More information about the Methods mailing list