Dual Vector backbones - cloning problems? (Feb/14/2006 )
I'm generating a vector from PCR-extended seperate fragments with long "fusion" overhangs.
I want to TA clone a vector fragment that I've produced by PCR, and I want to know what people's thoughts were as to the possible results of this?
It would create a vector with AmpR, KanR, two F1 single stranded DNA replication origins, and two pUC plasmid replication origins.
Are two pUC (or F1) rep origins deleterious to the vector? What do you reckon my chances are that if I TA cloned in a vector backbone into a TA vector, that I'd get what I'm after? I need it because I need to cut the ends of my PCR-produced vector fragment, but having the extra DNA sequence there from the other vector will give me both an indication that digestion has occurred, and a large amount of DNA to work with (since PCR product quantity is low) because I'll (hopefully) be able to prep-it.
i think that more than one origin of replication is not viable for the plasmid.