Ligation

richard at gpu.srv.ualberta.ca richard at gpu.srv.ualberta.ca
Wed Jun 18 20:35:49 EST 1997


Dear Robin, 

Your first stratigy usually doesn't work. But your 2nd one should be
working.

I guess the reason may be one of the following:
 
    1. What unmatched enzyme are you using? Klenow usually doensn't blunt
       3'-overhanging ends. If your unmatched ends are
       3'protruding(eg,KpnI), try T4 DNA polymerase which works for both
       5' and 3'.
        
    2. You maybe used too much Klenow or incubate too long time(15*C,
       15min is ok), this will result in the production of 3'-recessed
       ends
   
Hope this helps

Richard



Robin Young <internetalias at mayo.edu> wrote:
: Hi, there, I got some problems in ligating two fragments
: with only one matched cohensive ends. what I am doing
: is just put the two fragmnets together and add the T4 ligase 
: and then they should ligate to a linear fragment and I add Klenow
: to the reaction in order to blunt-ended the two unmatchends and add
: T4 ligase again to let the linear fragment self ligated to form the circle 
: plasmid. but this stratage doesn't  work. then I try another stratege, that
: is to isolate the fragments individualy by cutting with the unmatched enzyme
: and later using the Klenow to blunt-ended them and then cut with the
common(matched)
: enzyme and recover them from the gel. then do the normal ligation. but
this still doesn't 
: work. I don't know what is the problem, Is there anybody who has the
experience for this?
: I really appreciate the help.

: thanks. 

: Robin  




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