5 minutes ligation

Burkhard Hassel bhassel at orion.rz.mdc-berlin.de
Fri Nov 24 12:40:07 EST 1995


In article <49046a$fv8 at epervier.CC.UMontreal.CA>
Daniel Durocher <DurochD at IRCM.UMontreal.CA> writes:

> Can somebody tell me what is in the Boehringer "Rapid DNA ligation kit". 
> Is it somekind of salt, cofactor or just a demystification of the O/N 
> ligation?

I think its dewoodooization.
In our lab we usualy do ligations from 5 min to over weekend at room
temperature. I once tested a blunt end reaction:
Example of a ligation with different reaction times:
20 ng of pBLUE * EcoRV * CIAP (3kb)
30 ng of a 1,2 kb EcoRV Fragnment
ligation buffer         (= Maniatis 2nd issue page 1.68)
1 mM ATP       (end concentration)
1 u of T4 DNA Ligase       (Gibco)
---------------------total volume 10 ul.
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)


Burkhard Hassel (bhassel at orion.rz.mdc-berlin.de)



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