two bands after fill-in

L. Ramamurthy PhD lr50916 at
Mon Nov 17 12:14:14 EST 1997

Mr. G. Anil Kumar wrote:
> Dear friends,
> I am facing a very funny problem. When I am using a particular DNA insert
> for fill-in, after the fill in I am seeing two bands in the gel. This is
> repeatedly happening with only this template though I rountinely do this
> fill in with different templates without any problems.
>Well, this could be due to a number of reasons:
1. Do you Phenol/choloroform treat the restricted DNA before you treat
it with
DNA polymerase (Klenow or T4?)?
If not, any remaining restriction enzyme cuts after further incubation
at 37C
.. of course this means that there is one more site for this enzyme than
2. The other reason is a bit more far-fetched.. but possible, someone
contaminated your DNA pol with a restriction enzyme?

I suggest the following measures, set two separate digests (both of
[you think] are unique), enz A and enz B.. cut to completion (O/N if
need be).. phenol/chcl3 extract, precipitate. Then set up the Klenow
reaction. So this time you have used the same DNA but different
see if the same "funny Problem" happens!

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