trouble cloning a G/C-rich fragment

Juergen Brosius brosius at
Mon Oct 12 05:00:15 EST 1998

In article <JWVK0JAY3GI2EwgE at>, "Dr. Duncan Clark"
<duncan at> wrote:

> In article < at>,
> Juergen Brosius <brosius at> 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.


                                          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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