LMP agarose gels run in TAE buffer (used to be: Purification from Low Melt)
bernard at elsie.nci.nih.gov
Tue Mar 1 00:28:47 EST 1994
In article <smrodems-280294222013 at f180-045.net.wisc.edu>, smrodems at students.wisc.edu (Steve Rodems) writes:
> In article <vernon.16.0009A61A at micro.uct.ac.za>, vernon at micro.uct.ac.za
> (Vernon Coyne) wrote:
> > This is something that I have always done, but not known the reason why it is
> > so. Could you please enlighten me as to the reason one should prepare LMP
> > agarose gels in TAE instead of TBE when purifying DNA fragments from the gel.
> > (I also always use TAE gels for Southern blotting to nylon membranes).
> I have heard that borate can cause DNA damage so that some people stay away
> from borate containing buffers when the want to use the DNA for further
> cloning. However, I have been using TBE recently with the GeneClean II
> system and have had no problems. For me its easier to use TBE because our
> lab makes it up in 20L carboys. Also, I believe TBE gives better
> resolution so I've stopped using TAE altogether.
> Steve "Some day I will get the hell out of Wisconsin" Rodems
> "Then I am here for the Lee family renioun, shur-wajo-shur"
Just another opinion....
I generally use TBE for DNA electrophoresis because of its greater buffering
capacity than TAE (you can run gels at much higher voltages than TAE). The
resultant gels are fine for Southern blots or for gel purification of
fragments for use eg. as probes for Northern blots. The one time you can't
use TBE is when you intend to gel purify a fragment for ligation as the
borate inhibits the ligase (I don't know if this means *all* ligases).
Then I use TAE and run the gel overnight at low voltage.
In summary, I don't think the borate damages DNA per se but it can
interfere with handling it later with some enzymes (random primer labelling
with Klenow works just fine after TBE purfication).
All the above has been verified experimentally but I'd welcome
corrections and clarfications...
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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