Ultrasonic ligations

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri Aug 13 20:43:00 EST 1993


-----------------vernon at micro.uct.ac.za writes --------------------------
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.
*********************************************************
 
Hello ligation gurus!
I just thought I would add my comments to the discussion about
temperature.  I read some where,(one of the company fliers like FOCUS?),awhile
 ago
 that the half life of T4 DNA ligase activity was longer at reduced
temperatures
like 12 - 16 degrees.  Some one actually had some data showing the decline in
activity over time.  This is the reason I do all my ligations at this
temperature
  Even blunt end ligations.  I would just like to add when ever I have been
rushed to get a clone I never get the same number of colonies out of ligation,
sticky or blunt, if I only do the ligation a few hours on the bench.  The best
efficiency of transformable products always comes from the ligation overnight
at 12 - 16 degrees. (Sticky or blunt)  This may be moot if your clone is found
 in
in both sets of ligation products, but I find there is comfort in large
numbers
of transformants, like a few hundred, versus just 10 or so colonies from the
quicky ligation (especially blunts).  I should mention that I have been
following this discussion
on the Ultra sonic ligation and will probably try it. Anything to cut the time
of constructing plasmids!  Another thing that I have noticed that someone did
elude to is the unit definition of T4 DNA ligase.  Not all T4 ligase units are
created equal.  Most companies have expressed them in Weiss units (the
exchange
of nmoles of 32P from PP into Norit adsorbable substance) but others
like NEB still use there units based on cohesive end units, which according to
 
them is about 1/67 of a Weiss unit.  I mention this because in my hands blunts
go best with 5 - 8 Weiss units of T4 DNA ligase with a high concentration of
ends. For the novices out there check the unit definition on your ligase
preps,
especially if you are doing blunt end ligations.
Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________
 
 



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