B.M.s 5 minute ligations?

David Micklem drm21 at mole.bio.cam.ac.uk
Fri Oct 21 08:01:51 EST 1994


In article <DBOYD-191094143812 at nazar.dent.umanitoba.ca>,
DBOYD at ccu.umanitoba.ca (DAB) wrote:

> Boehringer Mannheim has a rapid ligation kit that they claim gives
> efficient ligations in 5 minutes; thats right only 5 minutes !!! Has
> anybody used it yet and does it work? Whats the secret ingredient?
> 
> It was the DNA that made me this way.
> 
> DAB
> University of Manitoba 
> Winnipeg, Canada

Probably nothing!  I routinely do sticky end ligations at room temperature
for 15 minutes, though I generally leave blunt end ones around for an hour
or so.  Works really well, even for complex ligations (eg 4 fragment).

Its instructive to try ligating MW markers (eg lambda HindIII) together
for short periods and then running them on a gel - most of the DNA ends up
at very high molecular weight very very quickly! (Unless of course you use
phosphatased markers by accident, but thats another story....)

(FWIW I use NEB ligase in their standard buffer.  I dont work for them, I
just like their enzymes)

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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Tel: [+44] (0)223 334129       Email:drm21 at mole.bio.cam.ac.uk
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