Ligating large fragments into pBluescript

Jeff "Newt" Sekelsky fznewt at mailbox.ucdavis.edu
Sun May 11 17:45:50 EST 1997


> At 14:41 5/9/97 +0000, Mark Leyland wrote:
> 
> >Has anybody got any experience of cloning quite large fragments of DNA
> >into plasmid vectors?
> >I'm trying to ligate fragments of 12kB and 18kb into pBluescript and
> >just wondered whether I was simply wasting my time or if it was possible
> >after all.
> >Any comments, tips or advice welcome.
> 
It might help to do the ligation in a larger volume.  With small inserts,
the first end ligating to vector is a bi-molecular reaction, but the
second end ligating to the other end of the vector is more like an
intramolecular reaction.  But with large inserts this is not true, so a
more dilute ligation reaction helps prevent getting concatamers.  Or you
can do a short ligation (5') in a smaller volume, then dilute to favor
circularization.

Also, as someone mentioned, larger plasmids transform much less
efficiently, so you may need to take extra steps to prevent recircularized
vector from being present.  Even with directional cloning, if the fragment
is very large (>15 kb), I often phosphatase the double-cut vector and gel
purify it.  Then I cut the ligation mixture with an enzyme that shouldn't
be present in the vector+insert products, but will cut empty vectors.

One last note:  as always, you can't rely on blue/white completely.  I
once had a 14 kb insert that gave blue colonies.

JS




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