DNA recovery from agarose gels-NA45

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Jul 18 14:55:31 EST 1994


    Several people have asked me for this protocol, so here it is. This is
(IMHO) the best way to recover DNA from gels. The membrane is Schleicher &
Schuell (Keene, NH 1-800-245-4024) brand NA45 (DEAE-cat #23410), and the
protocol is from the instructions included in the package. Since S&S gives
the protocol, I will only summarize it here; if you want to try it, order
the membrane and you will get the complete protocol.
    The basic idea is to run your DNA sample on a 'regular' gel (any way you
like) to separate the fragment of interest, and then make a slit with a
clean razor blade in front of the band to be recovered. (If there is another
band close behind this one, you need to make a slit in front of the second
band also, so you can stop it from eluting into the band of interest.) Place
a small strip of NA45 membrane into the slit, making it a little larger than
the band, so that the entire band can be caught by the membrane, then turn
on the power and run the gel for another five to ten minutes. Remove the gel
to a UV transilluminator (long wavelength!) to view the bands. The band of
interest should now be bound to the membrane, and none should be left in the
gel. Do not let the membrane dry at this stage. Remove the membrane strip
with forceps and place into a microfuge tube for washing. Wash two or three
times with NET buffer (150 mM NaCl, 0.1 mM EDTA, 20 mM Tris pH=8.0). At this
point, the membrane may be stored wet, or the DNA may be eluted.
    To elute the DNA, use HI-Salt NET (1 M NaCl, 0.1 mM EDTA, 20 mM Tris
pH=8.0), and incubate at 55-60C for 10-20 min., with frequent vortexing. For
higher yields, elute twice, using about 50-100 uL each time. Precipitate
with 2.5 volumes of EtOH, then re-precipitate with sodium acetate to remove
the NaCL (if necessary). Recovery is usually greater than 50-90% using this
method, and recovery can be monitored using the ethidium bromide
fluorescence as a guide. S&S claims this method is quantitative up to about
7ug/cm2 for DNAs less than 7kb. Good luck!
                Tracy
    
Tracy Aquilla, Post-doctoral Research Associate
Department of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu



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