Ligation in 5 min!

phillip buckhaults buckhaults at bscr.cc.uga.edu
Tue Jul 11 17:09:16 EST 1995


In article <drm21-1007952125070001 at drm-mac1.welc.cam.ac.uk>,
drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:

> oshiro_G at defiance.hsc.colorado.edu (Guy Oshiro) wrote:
> > Oops, looks like I have to rescind a little of my last post.
> > Seems that someone did do some quantitative transformations to test
> > ligation efficency.
> > 
> > >ligated at room temp,
> > >trafo 2 ul of ligation after
> > >10 min ---> 222 colonies (2/5 of transformation plated)
> > >35 min ---> 377 col.     (2/5 of transformation plated)
> > >73 min ---> 624 col.     (2/5 of transformation plated)
> > >22 h   ---> 680 col.     (2/5 of transformation plated)
> > >testliga without insert -> 22 col. (after 22 h)
> > 
> > 
> Yes, well, you'll notice that the difference between 73 minutes and
> overnight is very small (probably not significant, since this appears to
> be a single data point not an average).  I also think that this post was
> referring to a BLUNT end ligation, which takes longer in my experience.
> 
> I'm with you Guy - I've always done sticky-end ligations at room
> temperature for 15 minutes, and blunts for 1 hour.  Works very well, and
> saves a WHOLE DAY! No special kit required.
> 
> Checking ligated lamda HindIII (or similar )molecular weight markers on a
> gel after different times of ligation is very instructive...
> 
> Cheers, 
> 
> David
> 




dudes, 

if we keep cutting the overnight incubations out of molecular biology
there will be no more times for sleeping!  i liked it better when i
thought you had to let the ligation go overnight!
Phillip Buckhaults
Department of Biochemistry and Molecular Biology and
Complex Carbohydrate Research Center
Life Science Building
University of Georgia
Athens Georgia 30602 (706) 542-1701
________________________________________________________________________________

               "Let us not confuse effort with achievement" 



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