LMP agarose gels run in TAE buffer (used to be: Purification from Low Melt)

David Micklem drm21 at mbuk.bio.cam.ac.uk
Tue Mar 1 08:17:06 EST 1994


>
>Hi there!,
>	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
>

Well, FWIW, I've always used TBE gels for gel purification.  I've never had any
problems ligating them (even in three or four fragment ligations) after
purifying by several methods. (eg GenecleanII, Qaiex, elution onto GF/C
followed by phenol/chlf).  I've also used several different ligases, although
NEB ligase is my favourite.

Usual disclaimers apply....

David Micklem (drm21 at mbuk.bio.cam.ac.uk)

Really must fix up that .sig file



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