strange transformation results

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Apr 26 13:11:39 EST 2002


Frank,

One can see this even without the "stuffer".  I always gel purify 
vectors cut with 2 enzymes to avoid having that bit of polylinker, 
but I still see that result of having fewer colonies on the 
vector+insert plate.  However, if you think about it, inserts 
themselves should behave similarly to the "stuffer" you're talking 
about.  That is, if you ligate two inserts together, the ends are no 
longer compatible to make a circular recombinant plasmid.

Mike.

>
>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...
>
>
>Frank

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