Ligation in 5 min!

David Micklem 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...

Cheers, 

David

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D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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