Ligation - blunt vs single base overhang

Pamela Norton pnorton at lac.jci.tju.edu
Fri Aug 21 13:10:49 EST 1998


In article <199808201654.KAA42220 at nestor.NMSU.Edu>, hroychow at NMSU.EDU
(Hiranya Roychowdhury) wrote:

> This thing gets interesting by the day. I don't know how many will agree
> with me here, but I believe (from personal experience) that the "difficulty
> in blunt-end cloning" is overstated to the point where people have started
> believing religiously in this "myth". If the vector and insert preparations
> are clean enough, and the conditions are appropriate, blunt ligation is
> little different than sticky-end ligation. Hence, I would suggest that,
> unless you have extra time and money, to "stick" to your blunt fragments and
> to use a blunt-cut vector. You should get a good library even w/o having to
> "create" sticky ends.
> 
> Hiranya.
> 
> 
> Dr. Hiranya Sankar Roychowdhury
> Plant Genetic Engineering Lab.
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu

Hiranya,

     While I agree that excessive paranoia about blunt ended ligations is
unnecessary, they are not the same as sticky end ligations.* The ligase is
less efficient at the former than the latter. Solutions include adding more
enzyme and incubating the reaction for a longer time. Refer to any standard
methods manual and the information supplied with the enzyme.

     Pam Norton

    *However, to get back to the original poster's question, I think that
single base overhangs behave not much differently from blunt ends, but I
don't have a reference.

-- 
Pamela A. Norton, Ph.D.          Associate Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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