We do routinely out agarose gel electrophoresis with Lithium borate buffer instead of using the classics TAE or TBE (reference: Brody, JR, Calhoun, ES, Gallmeier, E, Creavalle, TD, Kern, SE (2004): Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37: 598-602)
We face at the moment problems with Qiagen's QIAquick Gel Extraction Kit, having only very low yields (around 0.5 - 2 ng/uL) but "fat" bands, and we thought the LiBo buffer is a possible source of the problems. Qiagen had no idea when we asked them. And of course we'd like to avoid to go back to the classic buffers. We use standard gel chamber, low-melt agarose and use a NanoDrop for quantification.
Does anybody used kits for DNA gel extraction with this buffer and experienced problems or is it possible without any?
Thanks for any input.
I've never tried it with LiBorate, but NaBorate buffer worked just fine with the Qiagen gel extraction kits. Or, at least as well as gel-extraction kits ever work. I've often had low yield even when using the conventional buffers.
We have recently found a way how to increase concentration after gel extract. We do 50 ul digest reaction. After that we have to gel purify DNA for ligation. What we do is that we run two lanes of the digest reaction on a gel. After that, we cut both gel lanes and try to excise minimum of gel. The point is that, we have in our tube maximum amount of DNA by loading all DNA after digest. Finally, we add only 30 or 20 ul of elution buffer. These steps have helped to increase concentration up to 10 or even to 20 ng/ul. However, gel purification did not help me with ligation. My ligation did not grow at all. Then I have found out that while gel purification almost all DNA can be ruined under UV. Hope it helps!
By the way, we use Invitrogen gel extraction kit.
Thanks for the ideas...DNA extraction out of gels seems still an effort after all these years...we'll try a kit from another company and then back to TAE if nothing works.
Some people use glass capillaries instead of razor blades to excise bands with as little gel as possible. You're already using low-melt agarose which should help
If you must use UV light, run a small amount of the digest in one lane as a marker, and the balance in a neighboring lane. Cut the gel between lanes and expose only the marker lane to UV. Cut the band and patch the lanes back together in the absence of UV on a clean surface. Cut out the non-exposed region of the sister lane.
I usually cut gel slices directly on UV at low intensity. It takes about 30 seconds to cut one slice of gel.
You can try to wait 2-3 minutes after adding elution buffer to QIAquick column, and only then start centrifuging.
Incubating the melting step in a thermo-shaker with vigorous shaking also helps a lot. Or vortex frequently and a lot and extend the melting step. As mentioned above - keep the amount of agarose as low as possible. NEB´s gel-extraction kit may also help, elution volumes can be as low as 6 uL. Else, eth-OH precipitate after the qiagen kit and redissolve in 5-10 uL...with the risk of losing your pellet ;-)
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