CMV promoter leaky in E.Coli?

b.schonhoft schonhoft.2 at osu.edu
Fri Aug 25 09:04:23 EST 1995


We are trying to subclone our gene into Clonetech's GFP vectors as a fusion
with GFP.  These plasmids are Kan^r/ Neo^r (under SV40 P) and have a pUC &
SV40 ori.  The IE promoter of CMV drives expression of gene fusions with
GFP.

So, here's my problem:  I use standard subcloning procedures and transform
commercial cells - I've used Gibco's DH5a and Stratagene's  Ultracompetant 
MR2 Blue's ~ both of which have worked well in the past.  After
transformation I plate out on 
LB+Kanamycin @ 30ug/ml. Colonies take ~48 hours to come up and barely grow
in LB+Kan o/n cultures for miniprepping.
Vector alone control is O.K.  

I'm thinking the CMV promoter may be leaky and my gene is toxic to E.Coli? 
 But I have this gene in several vectors, under several promoters and have
never had this problem with "leakiness".  
If anyone can help with this, I'd really appreciate it.  Thanks.


Brad Schonhoft
OSU Molecular Genetics
bschonho at magnus.acs.ohio-state.edu



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