DNA recovery from agarose gels-NA45

Frances Hannan Zoology flh at mole.bio.cam.ac.uk
Tue Jul 19 05:50:26 EST 1994


aquilla at salus.med.uvm.edu (Tracy Aquilla) writes:

>    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.

I agree with Tracy that this is the best way to recover DNA from gels however
I have never seen a protocol included with the membrane. The method I follow
is basically the same as Tracy's but with a few minor differences including 
pretreatment of the membrane with 10mM EDTA for 5' then 0.5M NaOH for 10' 
followed by 6x2' rinses in distilled H20. My High and Low salt buffers contain
50mM Tris pH8 rather than 20mM Tris but other components are the same. The DNA
is eluted in 2x150ul high salt washes at 65oC. After elution I add 24ul of 
0.25% n-acrylamide to act as a carrier (see NAR 18, 378) then phenol extract 
using 300ul TE buffered phenol and spin for 15' at 4oC to remove any shards of
membrane. After a chloroform/IAA extraction the DNA is precipitated with 700ul
ethanol. This DNA works beuatifully for all subsequent manipulations and the
yield is close to 90% most times.


>    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
-- 
Frances Hannan                                       
BILMS, Zoology, Downing St, Cambridge, CB2 3EJ, UK   
Phone (0223)336663, FAX (0223)461954                 
flh at mole.bio.cam.ac.uk                               



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