From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: supercoiled DNA migration in agrose [TBE vs. TAE or TPE gels]
Date: 28 Dec 1992 19:26:27 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: slc12.ins.cwru.edu


In a previous article, djt2@po.CWRU.Edu (Dennis J. Templeton) says:

>
(sorry to follow up my own post, but I got a question via Email that might 
be of general interest)

{{deleted the earlier discussion}}

>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.
>

The question I got was (with name removed to protect privacy)

>Hello,
>
>     I saw your post and had a question regarding use of TPE as a buffer.
>My recollection is that phosphate buffers have a greater tendency to grow
>various bugs than tris buffers. How is the mixture? Do you take any
>special precautions? And why to you prefer TPE to TAE for agarose gels,
>better buffer capacity?(I use TAE, as I think TBE yields inferior looking
>gels, but that may be all in my imagination).
>

Part of our reasons for preferring TPE over TAE is historical, though I
really do prefer either to TBE.                                

One event was when I was learning the glass powder purification protocol. 
I found empirically that having a little phosphate in the binding reaction
reduced the amount of non-releasable binding by about 1/3 (from 15% to 10%)
 For a while I put 10 mM PO4 in the NaI, but now I just rely on the
phosphate in the running buffer.  It is a *small* difference, granted.

The other event was that my neighbor (from whom I got the recipe for TAE)
 used HCL to adjust the pH; I don't know if all of the recipes around do or
not.  Our gel rigs consistently corroded at one end, and we hypothesized
that the buffer was releasing Cl2 by elecrolysis.

The TAE stock was maximally 20x too. Note that there are certainly 
TAE formulations different from the one I was dissatisfied with.

We use a simple TPE recipe that I made up myself; it is very similar to 
published ones, though some call for NaPO4, and I can't see using ions
that do not buffer, so I use only tris base and phosphoric acid.  I
lowered the ionic strength (to increase the voltage without overheating) to
the limiting point that the buffer has not changed pH by the end of the
run.                                                 
                                                              
Here's the recipe:

1x              for 50x TPE (500 ml)

36 mM Tris(base)        109 gm
Phosphoric acid
  to pH 7.4             about 30 ml
1 mM EDTA               9.3 gm
          
I don't have any direct experience with contamination of this buffer.
We store this on the shelf and have kept it 6 months or more without any 
beasties.  I think the EDTA helps keep them down.  I'd expect acetate to be
more of a problem since it is a carbon source, though I've never heard of a
problem with 20x TAE.

good luck, 

dennis