radiolabelled supercoiled plasmid

daniel jacobs d.jacobs at unsw.edu.au
Wed Oct 2 19:04:04 EST 1996


In article <3251A3FA.998 at sickkids.on.ca>, Gail Otulakowski
<gotulak at sickkids.on.ca> wrote:

> I wish to label plasmid with 32P, but I can't use nick translation or 
> random priming as these will affect the conformation and I need to look 
> at supercoiled plasmid.  I think I will have to add radiolabelled 
> inorganic phosphate to bacteria grown in minimal media - so two questions 
> - has anyone grown E coli in M9 minimal for plasmid preps and will it 
> make my yield lousy?  Also, M9 is normally buffered with phosphate - I 
> can buffer with Tris instead but what are E.coli's minimal requirements 
> for phosphate so I know how much hot stuff to add?

Unless you want masses of highly radioactive waste it might be easier to
label the plasmid in vitro and supercoil it with DNA gyrase. Just my
opinion.



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