supercoiled DNA migration in agrose [TBE vs. TAE or TPE gels]
afc at gnv.ifas.ufl.edu
afc at gnv.ifas.ufl.edu
Wed Dec 30 10:04:59 EST 1992
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:
>>chai_z at wehi.edu.au wrote:
>>: Several batches of M13 RF DNA of mine have been cut by EcoRI or SmaI,
>>: etc. and then electrophoresized in agrose gel. Uncut DNA (supercoiled) of some
>>: batches migrated faster than the cut one (linear), while the others slower.
>>: Most likely, some batches of my DNA were nicked (open circular). Could any one
>>: tell me the correct agrose pattern of supercoiled, open circular and linear DNA?
>>: or tell me where to refer to?
>>: Thanks in advance.
>>: Zhonglin Chai
>>In most agarose gels (but _not_ always), supercoiled circular DNA
>>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.
> To clarify Victors remark, the buffer used in Agarose gels affects the
> migration of superhelical plasmid DNA, and the other forms to a lesser
> In TBE gels, form II (open circles) migrate slowest, and form I (CCC or
> superhelical or supercoiled) migrates slightly faster. Form III migrates
> fastest of all in TBE (that's full length linear DNA)
> In tris acetate, or in the Tris phosphate buffer we prefer, form II
> migrates slowest, with form III running just slightly ahead, and form I
> (CCC) running far ahead of that. The exact migration varies depending on
> the size of the plasmid and the agarose concentration; in our 0.7% gels in
> TPE, a 3 kb plasmid (supercoil, CCC) migrates near the 1.6 kb band from the
> 1 kb ladder.
> We have never compared the migration in TBE and TPE directly, as it's hard
> to run adjacent lanes of a gel in different buffers!
> An added note, I would recommend saving your TBE for the acrylamide gels;
> TPE can be made up at 50x and allows your gel pieces to dissolve in NaI for
> the glass powder elution protocol.
> good luck
To clarify the clarification:
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
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.
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