supercoiled DNA migration in agrose [TBE vs. TAE or TPE gels]

Darren Natale camdna at ubvmsb.cc.buffalo.edu
Mon Jan 4 12:04:00 EST 1993


In article <1992Dec30.100500.2007 at gnv.ifas.ufl.edu>, afc at gnv.ifas.ufl.edu writes...
>In article <1hn8b6INN5ee at usenet.INS.CWRU.Edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>> 
>> In a previous article, vjongene at isrec-sun1.unil.ch (Victor Jongeneel) says:
>>>migrates fastest, followed by linear and nicked circles.  Linear does
>>>not always separate from supercoiled, nicked circles usually do.  This
>>>does of course not exclude the presence of miniphage (see other
>>>response in thread).  The best way to find out is to start
>>>with freshly purified (CsCl/EtBr) supercoiled DNA and treat it so as
>>>to make linears and nicked circles.
>>>
>> 
>The relative mobilities of the three different forms is affected by just
>about everything: buffer, agarose concentration, voltage, and probably
>moon phase.  In my hands, running TBE gels a la Maniatis, the following
>generalities hold.
> 
>Open circles run slowest.  A 15 kb mitochondrial molecule runs behind even
>uncut genomic DNA.
> 
>Supercoiled plasmids run at about the same mobility as linear DNA of 1/2
>the length.  I assume that this is because they are, in fact, linear 
>molecules of 1/2 the length of a linearized plasmid.  This is highly 
>dependent on the concentration of ethidium bromide in the gel.
> 
>There are lots of papers that discuss this issue rigorously.  I have one
>by Beverly (Nucleic Acids Res 16:925-939, 1988) that talks about pulse
>field gels, but also references the earlier literature.
> 

Another reference: Johnson and Grossman (1977) Biochemistry 16:4217-4224.
 This paper discusses affect of size, conformation, field strength, ionic
strength, and EthBr on migration.



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