Ligation in 5 min!
drm21 at mole.bio.cam.ac.uk
Mon Jul 10 16:25:07 EST 1995
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...
Wellcome/CRC Institute, Time flies like an arrow...
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