5' filling with Klenow
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