Ligation with T4 ligase

[L M]

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