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

Dennis J. Templeton djt2 at po.CWRU.Edu
Mon Dec 28 11:00:38 EST 1992


In a previous article, vjongene at isrec-sun1.unil.ch (Victor Jongeneel) says:

>chai_z at wehi.edu.au wrote:
>
>: Hi,
>
>: 	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.
>
>Cheers,
>

To clarify Victors remark, the buffer used in Agarose gels affects the 
migration of superhelical plasmid DNA, and the other forms to a lesser 
extent. 

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

dennis
 



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