How do you obtain your DNA fragment? If it by PCR, you can use PCR-labeling and integrate radioactive nucleotides this way.

Thanks, I hadn't thought of that! Is it possible to generate a supercoiled plasmid labeled plasmid through PCR, though? I thought the product would be linearized?

No, I don't think that you can generate supercoiled plasmid without bacteria. Are you performing EMSA with entire plasmid? It may be problematic due to the DNA size.

Yes, labeled supercoiled DNA is what I need for EMSA in order to compare to similar studies with linear DNAs. I'll keep looking...

Thank you anyway!

I suspect that your problem is how to label your plasmid as it is being replicated in the actual bacteria- and whilst a simple 32P T4 kinase or klenow fill reaction will give you a nice hot probe with little contamination of your lab I suspect that you are really wanting to steer clear of putting alpha 32PdATP into a culture of bacteria and then doing a highly radioactive maxiprep! Have you considered doing something like labelling your plasmid/bacteria with BrDU? After performing you EMSA you could blot the gel and do a western with an anti BrdU antibody. I suspect this will be less radioactive than in vivo labelling, and the probe should last you ages. Just a thought!

good luck