Ultrasonic Ligation

vernon VERNON at za.ac.uct.micro
Fri Aug 13 03:37:10 EST 1993


> In article <CBMp10.Jo7 at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu (t
>  he End) writes:
> >Max,
> >
> >16C as a temperature for ligation represents a compromise of optimal
> >temperature for annealing of short single stranded overhangs and that
> >for ligase activity. Blunt end ligations are traditionaly done at
> >room temperature as the first consideration is not important.
>
> Hmm, somehow this doesn't make sense to me. The ligation reaction is
> dependent on DNA concentration and the Km for blunt end ligation is about
> 100 fold higher so why would you think that you need to reduce the temp for
> sticky ends? Unless the off rate of the appropriate ends touching is much
> greater than the on rate and reducing the temperature slows only the off rate I
> just don't see it. Even if that were true lowering the temp would just slow
> down the ligase anyway.
>
As far as I know, one uses a ligation temperature of 16oC when
ligating sticky ended fragments as this is a compromise between the
temp at which ligase is optimally active (25oC) and 4oC at which
complimentary base pairing between the DNA fragments is most stable.
One is able to do blunt end ligations at higher temps as
complimentary base pairing is not a factor.








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