strange transformation results

Frank O. Fackelmayer Frank at
Fri Apr 26 11:59:31 EST 2002

William Sun wrote:
> I have sometimes observed a strange phenomenon while subcloning and would
> like to get some input.  When I subclone a piece of DNA into a cloning
> vector (I usually subclone using two different restr. enzymes) and transform
> into bacteria, I include a negative control of just the cut vector without
> insert.  Once in a while, I would get more colonies on my negative control
> than experimental plate.  But when I screen colonies from my experimental
> plate, most of them are positive!  Can anyone offer an explanation?

Hi William,
This is a very strange but common observation. I was quite surprised
when I realized it for the first time. My consequence was to not do the
negative controls any more. This doesn´t answer the question, but saves
work :)

I am not sure as to the reason, but I have the feeling it has to do with
the small stuffer you remove from the mcs in the vector. When there is
an excess of insert, the stuffer ligates to some of the insert molecules
(creating a fragment with two identical ends that does not clone into
the vector and is therefore "lost"), and you get positive clones (vector
+ insert). When there is no insert, as in the case of your negative
control, the stuffer ligates to the vector and rebuilds the original
vector. This is quite efficient and can create many colonies.

As I said: These are my thoughts, and I may be completely wrong. Anyway
it is plausible enough for me...


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