subcloning problem

cw via methods%40net.bio.net (by quetzales33 from gmail.com)
Thu Jan 21 09:34:34 EST 2010


Hi,

I recently encountered some problem with my subcloning - I PCR the CDS
of mCherry and Venus and inserted them into a 7kb vector to replace
the EGFP. The insertion is 5' to a loxP-Cre-loxP casette, and the
molar ratio of insert:vector=3:1. The PCR products were first TOPO
cloned then released by RE digest (PacI-NotI) and then gel purified
with the Qiagen kit. I then did the ligation 10 min @ RT using the NEB
T4 ligase....and transformed into Invitrogen's OneShot TOP10. I got
several clones for mCherry without any problem, but for the Venus
subcloning all the clones I picked were showing extensive level of
rearrangement/recombination.......does anyone know what might be going
wrong in my hands?

cw


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