Subcloning: Loosing my favorite gene

[Peter Ellis]

Allan wrote:
> Hi Peter,
>
> Thanks for your input.  I don't think I have explained myself well
> enough.
>
>> This cut site is in the gene insert, not the vector.
>
> My gene at this point is in pUC19 yielding pMyfavoritegene (pMFG).  I
> cut it with SgrAI to linearize and dephosphorylate and gel purify only
> the resultant 6.3 KB band.  At this point I don't know how a
> supercoiled pUC19 can migrate at 6.3 KB with the linearize pMFG.

It quite probably doesn't.  There could easily be only a tiny amount there, 
below the level of detection on a gel.  Just because you don't see a band 
doesn't mean it's not there.

Then, when your ligation fails, the only thing in the mix that's capable of 
generating transformants is the uncut, uninserted vector.

Peter