recover DNA from gels using agarase

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Thu May 6 19:06:34 EST 1993


In article <2109 at rwja.umdnj.edu>, bawagan at rwja.umdnj.edu (Hinayana Bawagan) writes:
> 
>     According to BM Biochemica March 93, recovery of DNA from low melting point agarose is about 80% efficient and is non-interfering with ligation reactions.  Does anyone have any experience with this method?  I am having problems with    glass milk method.
>     Thank you.
I was so late reading this posting that I thought, by now, someone might have
mentioned my 2 cents' worth, but they haven't so here goes:
The GELase agarase from Epicentre Technologies works very well for me in one
particular instance.  That is when I want to slice a particular band out of a
gel, cut it with another restriction enzyme, and run it on another gel.  This
happens if you want to verify that a particular site is within a particular
band.  In this case, you run your first restriction digest on SeaPlaque or
NuSieve in TAE buffer (I chill both the gel and the 1X buffer, so I can run it
fast at room temp, just like for TBE).  Then, you slice out the band of
interest and dilute it as in the GELase protocol, make it 1X in restriction
buffer and add the second enzyme and GELase enzyme simultaneously.  After the
GELase and restriction are complete (about 2 hours at 37C), you just add 2
volumes of cold ethanol and put on ice for 10 min.  Microfuge at 12,500 Xg for
15 min, pour off supe, and vacuum dry the pellet.  Now, you are ready to go on
the second gel.  I guess there are columns or some such gizmo that would also
remove the agarose, but since you are planning a second digest anyway...
AGAIN, I will just say that I do all of my ligations with the gel present by
the method of Crouse, et. al and that SeaPlaque and NuSieve do not interfere
with ligation.
Another good and fast way to rid your DNA of agarose is to extract the melted
slice with Tris-saturated phenol which has been pre-warmed to 65C.  I just
aliquot a small amount of the phenol into a microfuge tube under a fume hood,
cap it and put it in the 65C water bath with the tube which contains the gel
slice.  After melting the slice for 10 min, the phenol is warm.  Then, I just
add an equal volume of phenol and vortex vigorously.  After microfuging for 2
min to separate phases, you can see a whitish interface that has the agarose. 
I remove the upper aqueous phase to a new tube and then back-extract the
organic phase with another equal volume of warm TE.  After combining the 2
aqueous phases, I just precipitate with 2 volumes of cold ethanol on ice for 10
min and microfuge.  I have not calculated the yield, but I can tell that it is
very good.  This method does not require multiple phenol extractions or even a
single chloroform extraction.  If the volume of the 2 aqueous phases is too
large, all you have to do is butanol extract to reduce the volume before
precipitating.
That's all!
Barbara Bugg
St. Jude Children's Research Hospital
Memphis, TN  USA



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