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DNA from Low Melt Gel Procedure

Amos H Heckendorf nestgrp at world.std.com
Wed Jul 6 15:40:03 EST 1994


Nucleobond AX: 
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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 
starting.)

(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.

Regards,
Amos Heckendorf (nestgrp at world.std.com) 





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