Purifying clean plasmid vectors for ligation

Steven Goldberg goldberg at bms.com
Tue Mar 12 11:20:31 EST 1996


In article <Do3oxL.MtM at uns.bris.ac.uk>, harry.witchel at bristol.ac.uk wrote:

> Salutations Molecular Soul-brothers and soul-sisters --
>         I have had mixed success purifying plasmid vectors which have been 
> linearized, treated with phosphatase, and gel purified.  I have run the 
> purifications side by side, so the fact that the results are mixed is very 
> confusing...
>        Thanks one and all,
>         Harry

I was frustrated for years using phosphatase for the reasons you
mentioned...I usually resort to flooding the vector with insert (10:1
insert:vector) and screening my colonies by PCR.  I normally get 10-20% of
the desired recombinant plasmid.  If I purify fragments I use GeneClean
which seems to clean everything up nicely.  Best of all, if you can try
Invitrogen's pZeroI vector (should get at least 90% recombinants).


Good luck,

Steve



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