Subcloning: Loosing my favorite gene
(by pjie2 from cam.ac.uk)
Wed Feb 14 05:18:48 EST 2007
> Hi Peter,
> Thanks for your input. I don't think I have explained myself well
>> 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.
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