unstable insert

Ricky Boernke ricky_boernke at gmx.net
Mon Apr 8 02:27:00 EST 2002


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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