About ligation

David Micklem drm21 at mole.bio.cam.ac.uk
Fri Apr 26 06:54:24 EST 1996


In article <4lou5r$6a at lal.interserv.net>, Robert Cowherd
<rcowherd at mail.med.upenn.edu> wrote:

<snip snip snip>
>
>I've been told that it's actually a compromise between ligase activity,
kinetics of blunt DNA ends coming together, and ATP (in the =
>buffer) stability.  All four components, again I'm told, need to be in
the right-place-at-the-right-time:  1) one blunt end with 2) =
>a second blunt end and 3) an ATP molecule to enable 4) ligase to function
on those blunt ends.
>
>Self (intramolecular) ligation of blunt-ended vector follows first order
kinetics (faster because the ends whip around and have grea=
>ter numbers of "fortuitous collisions" with its complementary end - high
"local concentration").  Intermolecular ligation of blunt e=
>nds follow second order kinetics (therefore blunt ligations are better in
concentrated "small" rections of 5 microliters or so - inc=
>reased concentration of ends = larger #'s of beneficial collisions
between ends).  Sticky ends however can anneal to each other then=
> ligate "at leisure."  For sticky ends 4 degrees promotes optimal
annealling (relatively stable annealled end/complexes) and more st=
>able (high) concentration of ATP so the Vmax of ligase doesn't have to be
reached to get adequate number of ligation events.  In blu=
>nt end ligations, you'd prefer the highest ATP, DNA ends and Vmax of
ligase that you could get.  15 degrees (the compromise) seems t=
>o accomplish that....  

This all sounds fine to me, except that NEB state that blunt end ligations
are improved by DECREASING the rATP concentration from 1mM to 50uM!

Also, as I mentioned before, someone posted data (shock! horror!) that
indicated that blunt end ligations were essentially complete after 1hr at
room temperature: ie very little difference in number of colonies produced
afterr 1hr vs overnight ligation.  What I can't remember is whether the
overnight ligation was at room temperature or 15 degrees... 

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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