restriction inhibition of transformation

Peter Wang plw at mrc-lmb.cam.ac.uk
Tue Dec 9 12:29:37 EST 1997


kyoung at mail.med.und.nodak.edu wrote:
> I am attempting to move a plasmid from E. coli to an environmental
> isolate of Salmonella typhimurium.  I can move the plasmid into the S.
> typhimurium strain TR5877, which is res(-)mod(+), and this is good enough
> to modify the plasmid so that it can be transformed into the normal
> laboratory S.t. LT2 strains.  However, we can NOT get the plasmid into
> the environmental isolate, having tried all the regular transformation
> methods and electroporation.  Thus, it seems to be a restriction problem.
> What is the simplest method for circumventing such a restriction problem?

Dear Kevin:
I don't know if this will work in your case, but since most restriction systems 
protect DNA by methylation, you might try to methylate some plasmid DNA using a cell 
extract from your environmental isolate in the presence of EDTA (to inhibit 
endonucleases) and S-adenosylmethionine, then transform with the hopefully 
methylated plasmid.

- Peter

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Peter Wang, M.D., Ph.D.
MRC Centre for Protein Engineering,
Hills Road, Cambridge, CB2 2QH, England

Tel (01223) 402104  (international calls +44-1223-402104)
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