5' filling with Klenow

Martin Kennedy mkennedy at chmeds.ac.nz
Mon Sep 12 19:46:05 EST 1994

In article <35289a$te at mark.ucdavis.edu>, ez003450 at chip.ucdavis.edu () writes:
> I am constructing a fusion  protein by ligating two genes together.
> The juncture between the hybrid gene consists of a filled in 5'overhang 
> ligated to a SmaI site generating an in frame fusion. The 5' overhang is 
> filled in with Klenow. My question is how reliable is the fill in 
> reaction? Will all 4 bases of the overhang be consistently filled in or 
> is there any chance of 1 or 2 bases being removed(or extra bases being 
> added) and screwing up the reading frame? Do I need to sequence the
> juncture to confirm all 4 bases were filled in resulting in an 
> in-frame fusion? 

I would definitely sequence the junction.  Don't forget that Klenow still 
has a 3'->5' nuclease activity, and it can remove part of the 5' overhang.  
Although the fill-in reaction normally gives the desired results, I have 
lost one or more base at such sites more than once.



NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750

More information about the Methods mailing list