Self religation of double-cut plasmid vectors

Ken Saville saville at umail.umd.edu
Wed Jan 4 15:03:20 EST 1995


n article <dbell.2.00421AE8 at julian.uwo.ca>, dbell at julian.uwo.ca
(mahmoud)
wrote:

> I was wondering in your hands, if you purify your double digested plasmid 
> vector (any plasmid vector), and run a self religation experiment, what sort 
> of transformation rate do you see?

>After gel-purifying the linear, double-digested plasmid, I've never seen
>the self-religated plasmid in the colony screens.

-- 
>Bernard Heymann
bheymann at bragg.bio.purdue.edu


Recently, I was trying to clone a ss oligo into pUC19 digested with
HinDIII and SacI, The experiment was designed so that clones containing
the oligo would restore a blue phenotype, while uncut vector would
yield white colonies (i.e I used a plasmid with an insert as the source
of the double cut vector).
Ligation of just the cut vector yielded 3 white (probably uncut vector)
an 1 blue.  with the oligo I got 1 white and 3 blues.  These blues were
sequence an found not to have the oligo.  Instead, the plasmid had
religated with AGCT at the junction.  This would be expected if the 3'
SacI tail was digested and the HinDIII Tail was blunt ligated, or vice
versa, (if that makes sense).  Antway, it is possible to get unexpected
ligation products from double cut vectors.

cheers,
ken



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