unstable insert

Peter Ianakiev ianakiev at genome.wi.mit.edu
Tue Apr 9 09:24:53 EST 2002


Hi Ricky

I think you should slow down the E. Coli growth - say at room temperature.
Some DNA are unstable in high copy number plasmids - as pUC etc., try to use
low copy number plasmids like the old pBR322, or better F episomal plasmids.

Good luck
Peter


Ricky Boernke wrote:

> 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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