pCRblunt

Ashok Aiyar aiyar at ebv.mimnet.northwestern.edu
Thu Jan 25 21:07:04 EST 2001


On Thu, 25 Jan 2001 14:47:25 +0100,
    Susanne Rohrer (@immv.unizh.ch) wrote:
>
>
>"K.Fushimi" wrote:
>
>> Hi all!
>>
>> My friends have trouble in preparation of pCRblunt plasmid vector which
>> contains the ccdB
>> gene.  They store pCRblunt transformed E. coli with glycerol.
>> But when they use the stock, they often get wrong plasmid.
>
>Is the strain you use recA minus? Could your plasmid have undergone
>rearrangements?

ccdB is in fact toxic to cells.  Plasmids that express ccdB, such 
as pCR-Blunt that lacks any inserts, can only be propagated in
F' strains that express ccdA, or any strain that expresses ccdA.

The rearrangements in the plasmid probably inactivate expression
of ccdB and are therefore selected for.  We have propagated pCR-Blunt
in DH5alphaF', and MC1061/p3.

Ashok

-- 
Ashok Aiyar                                   a-aiyar at northwestern.edu  
Department of Microbiology-Immunology           office: (312) 503-2524
303 E. Chicago Avenue, WARD 4-123                  lab: (312) 503-2542
Northwestern University, Chicago, IL 60611         fax: (312) 503-1339






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