trouble cloning a G/C-rich fragment
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.
> > 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.
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.
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