trouble cloning a G/C-rich fragment

Juergen Brosius brosius at uni-muenster.de
Mon Oct 12 05:00:15 EST 1998


In article <JWVK0JAY3GI2EwgE at genesys.demon.co.uk>, "Dr. Duncan Clark"
<duncan at genesys.demon.co.uk> wrote:

> In article <RNA.world-1010981558200001 at expmac19.uni-muenster.de>,
> Juergen Brosius <brosius at uni-muenster.de> writes
> >After mutagenizing a fragment (about 2 kb - several point mutations were
> >made) of the HERG gene tried to clone it back into original vector
> >(pSP64/full-length HERG/A-tail of about 30 nt at 3' end of HERG).  While
> >the original vector appears to be stable we obtained only deletions of
> >various lengths.  Tried different E. coli strains including SURE.  Any
> >suggestions would be appreciated.
> >Regards,
> >          Juergen
> >
> 
> Can you switch off the background level of expression even more 'cos it
> sounds like your new mutations have conferred a lethality to the
> expressed protein that wasn't there before. Try a host with lacIQ or a
> host with same on a compatible plasmid etc.
> 
> Duncan
> -- 

Thanks for the idea.  Most of the hosts we tried were Iq.  Furthermore,
mutation is not complete, i.e. there is a certain percentage of WT. 
Therefore, we should have obtained some full length WT clones and my guess
is that the base composition of the fragment may be the culprit.

Juergen

-- 
                                          usual disclaimers
Juergen Brosius
Inst. f. eXp. Pathol.
University of Muenster
Von-Esmarch-Str. 56
D-48149 Muenster, Germany

Fax: +49  251  835 8512
Tel: +49  251  835 8511



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