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Ultrasonic Ligation

Michael G. Kurilla mgk2r at Virginia.EDU
Fri Aug 13 08:39:43 EST 1993


drmax at casbah.acns.nwu.edu  writes:
> 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. 
> 
While the reaction is dependent on DNA concentration I think
increasing the amount of DNA is bad idea.  Our typical ligation
follow the old BRL Focus articles of 20fm of vector and 60fm of
insert (whether blunt or sticky).  Even for large vectors (8kb)
this is about 100ng.  The problem of using too much DNA is that
at high enough levels, linear DNA can transform cells.  I think
that this can be a problem when lots of DNA is used and the
background of parental vector is high.  Most people assume that
the vector is either not purified away from uncut or the
phosphatase didn't go to completion.  I've seen people ligate
1ug and use 1/10 for the transformation; that 's just too much
DNA.

> 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.
 
For chemical transformations we used to dilute the reaction 1:5
with water and then transform with 1ul.  Since we began using
electroporation, we still dilute 1:5 but plate the
transformation 10% on 1 plate and the remaining 90% on a
second.  Most of the time we get dozens to hundreds on the 10%
plate.  

> I remember reading somewhere that 10% peg increases the efficiency of blunt
> ligations but I've never had occasion to try it.
> 
We typically do 10ul ligations using the BRL ligation buffer
with NEB ligase.  I have tested the ligases side by side with
their own buffers and crossed.  The NEB ligase with BRL buffer
(which has PEG) is the best combination.  Incidentally BRL will
sell just the buffer if you ask.  We have also tried Bio101 new
blunt end ligation magic and were not impressed.  For sticky
end ligations we routinely go 2 - 4 hours at room temperature.

> >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
> 
---------------------------------------

Michael Kurilla
mgk2r at virginia.edu



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