Paul N Hengen
pnh at fcsparc6.ncifcrf.gov
Fri Aug 13 11:25:45 EST 1993
In article <1993Aug13.060505.13012 at gserv1.dl.ac.uk> vernon at za.ac.uct.micro writes:
>> In article <CBMp10.Jo7 at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu
>>> 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.
Yes, the time interval of the juxtaposition of the 5' P to the 3' OH for blunt
ends is much less than for cohesive ends. Lowering the temperature of cohesive
end ligations also keeps the ends together for a longer period of time. For an
excellent explanation, see pages 81-88 of
Rodriguez, R. L., and R. C. Tait. 1983. Recombinant DNA techniques: An
introduction. Benjamin/Cummings Publishing Co., Inc. Menlo Park, CA.
It's also mentioned in the book chapter cited above that blunt-end ligations
may require two molecules of ligase - one to hold on to the ends and the other
to ligate them. Does anyone know if this is really true? I don't think it is
possible for them to occupy the same site. Perhaps one molecule binds to each
end and then they find each other to perform the reaction? Has this been
> 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.
I like to add the sticky-ended DNA fragments together at 30-35 C, mix, and
then place on ice for about 5 minutes to anneal the ends. I then add ligase
and allow the reaction to come up to room temperature on the bench over
lunchtime. If it's the end of the day, I'll put it at 16 C overnight. BTW,
if you're using the commercial supply of concentrated buffer, it most likely
has PEG in it. I add PEG to my own ligase buffer as well.
Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
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