ligation of incompatible sticky ends ???

John Ladasky ladasky at leland.Stanford.EDU
Sat Jun 14 19:36:21 EST 1997


In article <Pine.A41.3.95.970613200922.51726H-100000 at aix1.uottawa.ca>,
colossus... <s535290 at aix1.uottawa.ca> wrote:
>Here's a little stumper for you fine folks...
>
>The expression vector I'm cloning into will only accept NcoI/HindIII
>inserts. It already carries a reporter gene, which you can excise by
>NcoI/HindIII double digestion. I subsequently run such a digest, cut the
>vector band out (9 Kb - the excised reporter is 1.7 Kb) and gel purify it.
>
>Ligating into this doubly cut vector is being a pain in the ass on its
>own, but when I do a self ligation control, I manage to get a few
>colonies. I suppose there is a possibility of carryover of uncut vector
>during electrophoresis and gel purification...is this what's happening ? I
>always thought I was rather careful when doing gel purification of
>fragments and this is unnerving to say the least !!!

	It is quite likely that you are getting leftover uncut vector.  You
can of course confirm this yourself by either sizing the putative insert with
HindIII/NcoI digestion, or you could confirm by sequencing.  When I had a
double-cut ligation that wasn't working, the few colonies I obtained were
always uncut vector.

	Now, why might your ligation not be working?  Are you being careful
to examine and excise the cut vector from your gel under a long-wave UV 
lamp only?  Some sticky ends, and I think HindIII is one of them, are very
sensitive to short-wave UV light.  Are you using alkaline phosphatase in
your procedure?  (It's usually unnecessary for double-cut ligations.)  If 
yes, are you being careful to inactivate it before the ligation step?  Are
you sure that your insert is cut correctly?  Do you have enough overhanging
nucleotides at the ends of your insert so that the restriction enzymes can
cut?

	Good luck!

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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