Ligation with T4 ligase

Keith Rand rand at pelican.dbe.csiro.au
Thu May 29 04:34:05 EST 1997


Maybe the fragments in question both have HindIII at one end and Xba at
the other? Anyway, Richard points out a very useful method for joining
otherwise incompatible ends. I've used the method several times
successfully (although with T4 polymerase). For anyone doing this work I
can recommend the papers  describing how to do this:

Title 
       Cross index for improving cloning selectivity by partially
       filling in 5'-extensions of DNA produced by type II
       restriction endonucleases. 
Author 
       Korch_C 
Source 
       Nucleic Acids Res, 1987 Apr 24, 15:8, 3199-220 
Abstract 
       A cross index is presented for using the improved selectivity
       offered by the Hung and Wensink (Nucl. Acids Res. 12,
       1863-1874, 1984) method of partially filling in
       5'-extensions produced by type II restriction endonucleases.
       After this treatment, DNA fragments which normally cannot
       be ligated to one another, can be joined providing that
       complementary cohesive ends have been generated. The
       uses of this technique, which include the prevention of
       DNA fragments (both vector and insert) auto-annealing, are
       discussed.  






In article <5mg236$q24$1 at pulp.ucs.ualberta.ca>, lm11 at gpu3.srv.ualberta.ca
(L M) wrote:

> Dear ER,
> 
> Both Hind3 and Xba1 produce 4-base extensions, but only 2 bases are
> compatible to each other, i.e. GA (in Hind3) pairs with CT (in Xba1):
> 
> Hind3: 5'-A
>          -TTCGA-5'
> 
> Xha1:  5'-CTAGA-
>               T-5'
> 
> In this case, it's much more difficult to ligate them togather, because 
> the H-bond is very weak, but it's ligatable! 
> 
> Before you ligate them, you need to make a brief filling by adding
> dATP+dGTP+Klenow to Hind3 product, and adding dCTP+dTTP+Klenow to Xba1
> product. You need also to inactivate Klenow by heating before you combine
> them. By doing this I made a succeful cloning some time ago.
>   
> Good luck
> 
> Richard
> 
> 
> Estanislao Ramirez (eramirez at BITMED.MED.UCHILE.CL) wrote:
> : Dear Everybody:
> : I've got a problem with a ligation with T4 ligase.  I have purified two
> : DNA fragments from an agarose gel.  Each of the fragments has sticky
> : ends (cut with Hind III and Xba I) and therefor should be
> : complementary.  Is there any way to investigate the efficiency of a
> : ligation before attempting a transformation?  thanx
> : 
> : Alfredo Ramirez
> : School of Medicine
> : Universidad de Chile

-- 
Tired Khan,  Sydney Australia



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