Blunt ending??

Dom Spinella dspinella at chugaibio.com
Tue Jun 9 09:31:27 EST 1998


> Everyone else seems to recommend Klenow + dNTPs. For reasons outlined
> in (1) I would recommend T4 DNA polymerase + dNTPs for all routine
> blunt-ending. It works for me, at least...


Most people use Klenow for 5' overhangs, relying on the polymerase
activity to extend the 3' end to make a blunt end.  T4 pol has the same
activity and is certainly usable in this regard. For 3' overhangs, where
the 3'->5' exo activity is what's needed, T4 Pol is better, as Klenow
has much weaker exo activity. However, "in the old days" T4 pol was much
more expensive than Klenow and was therefore less used for 5' fill-ins
(where both enzymes work equally well).  In recent years the price
differntial has come down quite a bit, no doubt due to over-expression
of both enzymes in E. coli as the commercial source ($81/100 units for
T4 pol vs $53/100 units for Klenow in the latest B-M catalog). Given
that, I suppose it may make sense just to have T4 pol in the freezer and
use it for blunting both 3' and 5' overhangs.  Cheers.
--Dom Spinella



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