GATEWAY cloning of large PCR fragments

Ricky Boernke ricky_boernke at gmx.net
Fri Sep 3 08:54:55 EST 2004


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



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