Ligation in 5 min!
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...
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!
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