DNA recovery from TBE gel
bpmurray*STUFFER* at socrates.ucsf.edu
Wed Jun 24 21:24:37 EST 1998
<Pine.SOL.3.96.980624190755.5242A-100000 at ascc.artsci.wustl.edu>, Alex
Brands <abbrands at artsci.wustl.edu> wrote:
> Hello all,
> I'm doing a partial digest of a 10.5kb plasmid, and I need to gel purify
> the linear DNA from the uncut. On a .7% TBE gel, the linear and uncut
> bands are resolved nicely. However, when I ran the digest on a .7% TAE
> gel, the bands were not resolved.
> Now, I've always heard that you're supposed to do gel purification with
> TAE gels, although I don't know what the problem with TBE is.
> My questions are; What is the problem with isolating DNA from TBE gels?
> Is there some way around the problem?
> Is there some other solution to my problem?
Okay, my *personal* opinions/findings are as follows;
Gel purification in the presence of TBE is fine for most purposes
except when you intend to use the fragment in ligation. Borate
inhibits T4 DNA ligase so the efficiency goes waaaay down.
This being said I've had arguments with co-workers who are
happy ligating fragments after extraction from TBE gels. This could
partly be due to the means of extraction from the gel (eg. QIAex or
Wizard work but freeze/squeeze does not) or simply by using small
amounts of the DNA solution in a largeish reaction volume and
having a high efficiency ligation.
The bottom line is that a TBE gel is your best means of
purification so go with that and try and clean up the DNA if you
have to ligate.
I hope that something works,
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
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