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Dear Gel cutters:
We have tried the following and gotten near quantitative recoveries:
from Nucleobondª Technical Notes Number 5.1.4-1
Nucleobond is a trademark of Macherey-Nagel, Germany.
The Nest Group, Inc. 45 Valley Road, Southborough, MA 1-800-347-6378
Alternative Procedure for Using Nucleobondª AX Cartridges to Isolate
Double-Stranded DNA (>60bp) from Low Melting Point Agarose Gels
The use of low melting point (LMP) agarose gels has greatly facilitated
many procedures in molecular biology. This type of agarose, although
difficult to handle at concentrations less than 1.0%, has opened the door
to fairly quick ways of isolating nucleic acids from gels without the
need for electroelution, enzymes, or other methods. In fact, in some
cases, (for example, ligations and random primed oligo-labelling
procedures) the reactions can be done right in the LMP gel without the
requirement for prior purification.
A number of different procedures have been developed to take advantage of
the LMP quality to purify nucleic acids from these gels. These methods
include dissolving urea into a molten gel slice, freezing/thawing in
phenol, and filtration. Filters work very well when used in concert with
Nucleobond cartridges. A cellulose acetate filter removes most of the
agarose from a re-melted and diluted gel slice solution. The Nucleobond
AX purifies the nucleic acid from any other contaminants that may have
been in the agarose. (Note: please read the entire procedure before
(1)Excise the DNA gel slice in as small a piece as possible (the
volume should be on the order of 50-100ml).
(2)Re-melt the agarose by incubating at 65-70oC for 5-10 minutes and
immediately add 1ml of buffer N1 prewarmed to >35oC. Mix by
vortexing and incubate at 37oC until needed. (Alternatively, the gel
slice can be placed in the N1 buffer first and then heated to 70oC
for 5-10 minutes or as required. However, the lid of the tube should
be wrapped with parafilm to prevent any evaporation of the ethanol in the
buffer. Vortex and incubate at 37oC for 5-10 minutes before
loading. For larger fragments, greater than 150bp, the N2 buffer
can be substituted for N1 in this step.)
(3)Equilibrate an AX-20 cartridge with 1ml of the N1 buffer.
(Again, for larger fragments, buffer N2 can be used.)
(4)Filter the diluted gel slice through a 0.45mm cellulose acetate
disk while, at the same time, loading the sample onto the cartridge.
(5)Wash the cartridge with 3 x 1ml of buffer N1/N2 (1.5ml N1 + 1.5ml
N2). (For larger fragments, greater than 150bp, the N3 buffer can
be used for the wash steps. The salt concentration of the N3 buffer
will ensure greater purity, however, it is also high enough to
elute fragments smaller than 150bp and is not recommended in those cases.)
(6)Elute the DNA with 0.9ml of buffer N5 and precipitate with 0.8
volumes of isopropanol.
Amos Heckendorf (nestgrp at world.std.com)