duplicate ORI construct

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Mon Sep 25 12:37:52 EST 1995


>Hi netters,
>A friend of mine was asked to make the following construct: he is to ligate the
>linearized pCATbasic(from Promega) into another plasmid that contain a promoter
>that they want to study. In short the resulting construct would be a plasmid
>that consist of two "vectors" ligated together with the duplication of the ORI
>and the AMP gene and etc. What is the likelyhood of success for such a
>construct? I think they need the whole pCAT first for the CAT reporter and the
>rest of the plasmid as a stuffer fragment to study the expression of the
>neighbouring gene, something to do with spacial proximity and such.
>Any comments appreciated either posted here or at this address.
>Thanks,
>
>K. Chan
>IMB/HKU
 
I was told many times that plasmids with a duplicated ORI
should not survive. Then I did some subcloning from pBC SK 
(Cm-res, colE1 ori, Stratagene) into pBS SK (Amp-res, colE1 ori, same)
and found that the white colonies growing on amp plates contained
either pBS with insert either a single plasmid pBS+pBC.
The cloning  was done in RecA- cells, and cells containing pBS+pBC
were growing slower than those with insert and were double (amp+cm)
resistant.

Bottomline:
it might work in recombination deficient strains.

Best regards

Andrei Popov
      





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