Ligation with T4 ligase

L M lm11 at gpu3.srv.ualberta.ca
Tue May 27 20:59:37 EST 1997


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

Dear ER

Hd3 and Xb1 have compatible sticky ends, but not perfect, just partly. GA
(in Hd3) pairs with CT (in Xb1):

Hd3: 5'-A
     3'-TTCGA-5'

Xb1: 5'-CTAGA-3'
            T-5'

In this case it's very hard to ligate them together. before ligation you
need to make a brief filling reaction by adding dA, dG and Klenow to Hd3
product, and adding dT, dC and Klenow to Xb1 product. Also you need to
inactivate Klenow by heating before you combine them. By doing this I did
a successful similar cloining sometime ago.

Good luck

Richard




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