ultrasonic ligation

suter at de.d400.mpg.MPIZ-KOELN.VAX suter at de.d400.mpg.MPIZ-KOELN.VAX
Thu Aug 12 06:41:38 EST 1993


in a previous message, Max wrote:

+There seems to be lots of confusion out there about ligation protocols.
+Wildtype T4 ligase had inherent endonuclease activity and ligations were
+incubated at 16oC to block the endonuclease activity. This also slowed to
		      ???????????????????????????????		
+ligation reaction since the ligase likes something closer to roomtemp for
+maximal activity. The T4 ligase we use today is from a recombinant which no
+longer has the endonuclease activity. There is no reason to incubate at
+16oC anymore (unless for some reason you want to slow down the ligation
+reaction!).
+
+If you are very very concerned to get every last bit of your DNA ligated
+then maybe you might want to resort to longer incubations or ultrasonic
+cleaners but for run-of-the-mill stuff where you just need to get a
+particular fragment subcloned or a PCR product into a vector, a few minutes
+on the bench is sufficient.

i tested this hypothesis a few years back, and ligated stickies for 5', 
10' , 2 hours (at RT) and one reaction for 16 hours at 14C. The latter reaction
gave a relative 100 % clones, 5' and 10' at RT around 5% and 2 hours at RT
75%. 
I must admit that I actually added 0.015 Weiss units, whereas most (richer ?)
scientists will just add 0.5-1 microliter T4 ligase. i also didn't analyze
all clones for insert-uptake.
i think it is ill-advised to try to clone PCR products (which can be hell
to clone anyway) by short RT ligations. 


cheers, clemens



===============================================================================
Dr. Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
===============================================================================
	if this is victory, our hands are to small to hold it



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