Subcloning: Loosing my favorite gene

[Allan]

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.
Also, I intend to put my GFP into this SgrAI cut site.



> >
> What do you mean by a linearized gene?  Genes aren't naturally circular.  I
> presume you mean that you've cut the gene with SgrAI.
>


> You should ligate the GFP to your gene before cloning it into the plasmid.

The idea behind this project is to clone a flourescent tag inside the
gene.  The gene is cleaved at both the N and C termini so I can put
the gene downstream of GFP.  My source of GFP is clone I built from a
protein expression vector.  My pGFP is being cut with SgrAI,
electrophoresed and gel purfied.

If you're able to think of anything else I'd appreciate hearing it.

-A