cohesive and blunt ligation protocols

Claude Aflalo aflaloc at BGUMAIL.BGU.AC.IL
Tue May 23 05:40:08 EST 1995


On 22 May 1995, Joaquim Culi wrote:

> Dear Netters,
>  Have you ever tried to ligate a fragment with one compatible cohesive
> termini and another non compatible? I've been trying for two weeks and I
> haven4t found yet the correct way to do it. In my protocol I ligate the
> compatible cohesive termini first and then I try (unsuccessfully) to fill
> the other protuding termini with Klenow enzyme and a mixture of dNTP to
> make it blunt, and finally ligate again. 
>   I would be forever grateful if you would send me your protocols for such
> a ligation or others that would solve my problem.Thank you.
> 
> 
Joaquim,
If you got the first (cohesive ends) ligation right, you should perform 
the fill-in reaction with Klenow large sub-unit and the blunt ends 
ligation at **low** DNA concentration in order to prevent formation of 
oligomers.  
Hope this helps...

Claude Aflalo ###########################################################
Dept. of Life Sciences			Phones: Office 972-7-472118
Ben Gurion University of the Negev	        Lab    972-7-472119
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Beer Sheva 84105 Israel			email:  aflaloc at bgumail.bgu.ac.il




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