unstable insert

Michael O'Donohue michael.odonohue at univ-reims.fr
Thu Apr 11 01:46:09 EST 2002


Hi Ricky,

I was very interested in your post because I have exactly the same problem
with a synthetic gene that I have made. In my case, the codon usage is that
of B. subtilis (48% G-C) and I have no detectable repeats (direct, inverted
or otherwise).  I even tried TOPO cloning but this does not work. The only
suggestion I have is to use ABLE red or Yellow E. coli. These reduce plasmid
copy  number. However, I would really like to know what the actual cause of
DNA instability is. Nobody seems to talk about this problem although I am
sure that lots of people encounter such difficulties in cloning work.

Mike

Ricky Boernke <ricky_boernke at gmx.net> a écrit dans le message :
3CB14644.D5BBD65F at gmx.net...
> Dear all,
>
> we have made a synthetic gene in order to adapt it for plant codon
> preference. The company which actually made it already reported that
> the had difficulties to clone the insert into pUC and to propagate
> the plasmid in E. coli. But apparently they finally got it. However,
> we are now encountering serious problems when we try to clone
> the insert into other vectors. It is just 1.6 kb but we do not obtain
> any positive clones, neither after re-PCR of the fragment and ligation
> into pCR-Blunt nor after excision from the original plasmid and ligation
> into pQE. We have had some positive mini preps but after reculturing
> the plasmids were empty again. We have tried different E. coli strains
> such as SURE cells from Stratagene or Rosetta cells from Novagen to
> enhance insert stability but without any success.
> Does anybody have any idea about that?
>
> TIA
> Ricky





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