Linear plasmid for transfection - where should I cut the plasmid? (Oct/01/2007 )

Hi everyone.

I got an easy problem which I'm not able to find an answer myself. It shouldn't be difficult for some of the great experts around this site (I hope)

I want to linarize my plasmid (pTarget) in order to trasnfect it to create a stable cell line. Using supercoiled or circular DNA I haven't been able (three times). I'm using pTarget and my gene of interest is after the CMV/T7 promoters WHERE SHOULD i CUT THE PLASMID (pTARGET)? Is it ok after my gene? Will the Neomycin cassette be still working fine? It is likely that if I got neomycin resitant cell lines, they will express my gene of interest?

pTarget vector map in this link:
http://www.promega.com/figures/popup.asp?f...n+Vector+System

I was planning of trying tomorrow, so please give me some advice.

Thanks a lot.

Anywhere from the end of the poly A region at the end of Neo to the beginning of the CMV promoter region. You don't care about the amp resistance gene or the E. coli origin for the plassmid, so it doesn't matter where those are cut.

Thanks a lot. For the record, I'm gonna use ScaI which is within the AMP cassette and it doesn't cut my gene of interest.

One more thing, why cannot you use the multiple Restriction Site downstream of my gene? The Neo gene and their promoters will not be disrupted.

You might be able to use those sites. The linear fragment is supposed to be inserted, and you are constructing the fragment as a plasmid. The only thing that would happen with that site would be that the two sections (neo gene and your gene of interest) would be near the ends of the linear fragment. This might or might not be an issue. Mainly, you are attempting to avoid destroying the relationship between the genes and their promoters, or interrupting the genes or prmoters themselves.