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...
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