cloning problem...

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Jul 31 05:48:44 EST 2000


Hi Anne-Marie,
We also had this problem several times. We found no remedy, I´m afraid
to say. When we tried a troubleshooting, we tested virtually everything.
Nothing helped. The only correlation we found is that this problem only
occurs with expression clones, where the insert in behind a prokaryotic
promoter. We never saw anything like it with all other clonings. This
would suggests that the insert (i.e. the encoded protein...) is toxic to
the cell, but why then do these clones form colonies on plate after
transformation? 
Well, we thought that the growth conditions on plates are different from
those in liquid medium. In fact, when we re-streak the clones on plates,
they DO grow, so we made minipreps from bacteria streaked onto plates in
an area some 1cm in diameter. This gave enough DNA for analysis by
restriction digests. However, we were not able to persuade the positive
clones to grow in liquid medium... We finally gave up. Now, when we have
this problem (it rarely occurs, of course), we simply say it isn´t
possible to work with them.

I know that is no help, but you´re not alone.

Frank




"Dr. Roland Barten" wrote:
> 
> We have been trying to clone a human gene in to TA vector. We've got
> blue/white selection,  have screened the white ones directly by PCR, and
> then tried to grow up those with inserts - this is where the problem
> starts. On trying to grow up for mini-preps, nothing happens. There is
> absolutley no growth.
> We have tried different media and both Kan and Amp selections (as well
> as no antibiotics at all), but we just cannot get any of these colonies
> to grow in liquid media - except for once when it turned out that the
> insert had two base pair changes.
> One option would be phage - any other suggestions?
> 
> Thanks
> Anne-Marie Lucas






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