In article <CBMp10.Jo7 at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu (the 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.
Seems to me that the best way to improve the rate of ligation would be to
increase the concentration of DNA. I do my reactions in 5-10uls and use
only half the reaction in my transformation (for Hannahans - 1/10 for
electorporation). If I use more I get too many colonies on the plate. I
haven't had much occasion to do blunt ligations and I design my PCR primers
with restriction sites so perhaps you are right for those types of
ligations.
I remember reading somewhere that 10% peg increases the efficiency of blunt
ligations but I've never had occasion to try it.
>>There does not appear to be a "routine ligation" as different combinations
>of DNA fragments ligate with a widely different efficiencies. In approaching
>a new combination of DNA fragments, I find it necessary to strive for the
>highest possible ligation efficiency as difficult constructs are
>as common as theoretically "simple" ones. Several-fold differences
>in ligation efficiency can often make or break a plasmid construct
>in my experience.
Well I'll take the clone one day sooner if I can get it especially if the
reduction in efficiency is only 25% as one poster claimed. An easy way to
cover one's bases (pun intended) is to set up the ligation, pull out half
of it after an hr on the bench and transform with it. Put the other half at
16oC overnight. If you don't get enough colonies on your plate the next
morning then you can transform with the other half without losing any time.
For the person who made the big ????? under my comment about endonuclease
activity in wildtype T4 ligase. My understanding is that completely
purifying the bacteriophage T4 ligase wasn't possible and that there was a bT4
protein that attacks cytosine clusters left in the reagent.
Max
