Help! Direction of ligation

Pekka Rappu TUY prappu at finabo.abo.fi
Tue Sep 24 02:16:53 EST 1996


On 23 Sep 1996, Irina A. Libova wrote:

> Hi!
> I am ligating an insert into a vector, using HindiIII site on both 
> ends.  And I need a specific orientation.  But the problem is that on 
> ALL of my clones I get the reverse orientation compared to the one I 
> need, instead of getting a random 50:50 distribution of orientations.  
> Has anybode had the same problem, and what can I do to change the 
> orientation?
> 
> 

Hello Irina,

It seems to me that the host can't survive with the insert in the forward
orientation. The gene product can be lethal for the host at least as high
doses. Is the vector a high copy one? The host could tolerate fewer copies
of the insert. I recommend using either low-copy plasmid or low copy
strain (e.g. E. coli KE94). 


Pekka Rappu	Biochemistry, University of Turku
              	FIN-20014 Turku, Finland
              	++358-2-333 6856
	      	FAX ++358-2-333 6860
              	pekrappu at utu.fi
		pekka.rappu at utu.fi
              




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