Does DNA have to be supercoiled for transient protein expression? - (Feb/02/2012 )
I purified my DNA using a Qiagen Maxiprep kit, and I have two bands on my agarose/EtBr gel. I'm assuming that one is supercoiled, while the other may be linear. For transfection and expression of protein, is the only usable DNA the supercoiled portion?
Either will work, however supercoiled DNA transfects more efficiently than circular or linear DNA.
What could be happening that would cause my dna to be circular/linear, instead of supercoiled? How do I maximize the supercoiled form?
It is pretty much impossible to get a prep that is 100% supercoiled; there is almost always some degradation during extraction. Much of this degradation comes from shear forces in centrifugation steps and from chemical digestion during the alkaline step.
I agree with bob1. You will always have supercoiled DNA along with some nicked DNA.
DNA from maxi prep should give you a good percentage of supercoiled DNA. And be gentle.