how to separate the nicked from of a plasmid from the closed form?

Zophonias Oddur Jonsson zjons at rhi.hi.is
Tue Jun 7 19:57:33 EST 1994


In <1994Jun7.171123.1 at icrf.icnet.uk> t_rolden at icrf.icnet.uk writes:

>does anyone know a method to separate the nicked circular form of a plasmid
>from the covalently closed circular form?. I have a mix which contain 50% of
>each form, when I do a CsCl gradient to isolate the closed form I always have
>contamination with the open form. does anyone konw a method to separare them,
>different from the CsCl gradient?.

You can separate nicked plasmids from closed circular on Ethidium Bromide 
containing agarose gels.  50 ul EtdBr in E-buffer works fine.  The nicked form
runs a lot slower than closed plasmids under these conditions since it can 
bind a lot more of the dye.  You cold then simply cut out the bands and elute
the DNA.  I do however think that if you need large quantities of DNA a CsCl 
spin would be more practical.  If you don't overload the gradient and have 
enough EtdBr present I can't see why it shouldn't work.  Bauer and Viongrad 
did it back in the sixties and a lot of people have done it since then!!

>Many thanks,
>T. Roldan

Good luck
Zophonias O. Jonsson
University of Iceland





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