Generate Supercoil Plasmid - it just a thought... (Aug/15/2010 )
I just came across of this thought when I'm having an afternoon nap.
I know is possible for us to generate a linearized plasmid by doing inverse PCR.
From here, is it possible if we can make it in a form of supercoil?
PCR fragments can be ligated to form circular plasmids, which can be transformed. In the cell, topoisomerases will supercoil plasmids and maintain the proper level of coiling. The final ligation of virtually every cloning experiment ligates a linear to a circular fragment, which is then transformed. PCR fragments are no different than other DNA (modulo possible methylation).
in order to make supercoil plasmids synthetically without using a living host, not possible?
DNA gyrase will introduce negative supercoiling in buffer + ATP. See NEB product M0306. This will only work on un-nicked circular DNA fragments. Supercoiling can be reversed with Topoisomerase I.
why would you want to do this? for more robust plasmid storage or something?
if you're after better transformation efficiency, I remember reading that closed circular plasmid DNA transforms E. coli with higher efficiency than supercoiled plasmid DNA...
Thanks for your information and prompt reply.
Mycobacterial Madman: I was told supercoiled plasmid is good quality plasmid... that's the reason I'm looking into that...