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-labeling 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. In this guide two different procedures are presented: 1. Freezing/thawing in phenol and 2. Filtration. (Note: Please read the entire procedure before starting.)
If the DNA is to be purified from a regular, high-melting-point agarose gel, then an alternative product such as NucleoSpin Extract or NucleoTrap should be used. The latter kits consist of a silica membrane in a spin column format or loose silica resin, respectively. Gel dissolution and DNA binding is performed using chaotropic salts and elution is accomplished with a low ionic strength buffer such as TE. Please refer to the NucleoSpin and NucleoTrap Properties and Applications Guide for further details on these kits.
1. Excise the DNA gel slice in as small a piece as possible (the volume should be on the order of 50-100 ”l).
2. Re-melt the agarose by incubating at 65-70C for 5-10 minutes and immediately add 1 ml of buffer N2 prewarmed to >35C. Mix by vortexing and incubate at 37C until needed. (Alternatively, the gel slice can be placed in the N2 buffer first and then heated to 70C 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 37C for 5-10 minutes before loading. For smaller fragments, less than 100 bp, the N1 buffer should be substituted for N2 in this step.)
3. Equilibrate an AX-5 or an AX-20 cartridge with 1 ml of the N2 buffer. (Again, for smaller fragments, buffer N1 should be used.)
4. Filter the warm, diluted gel slice through a 0.45 ”m cellulose acetate disk while, at the same time, loading the sample onto the cartridge.
5. Wash the cartridge with 3 x 1 ml of buffer N3. (For smaller fragments, less than 100 bp, the N1/N2 (1.5 ml N1 + 1.5 ml N2) buffer should 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 100 bp and is not recommended in those cases.)
6. Elute dsDNA fragment with 0.9 ml buffer N5.
7. Precipitate the DNA by adding 0.8 volumes of iso-propanol and centrifuge at 12,000 x g for 10-15 minutes.
8. Wash the pellet very carefully with 70% ethanol, air dry briefly, and dissolve in an appropriate buffer for further manipulations.
Buffer solutions: Storage: N1 - 100 mM Tris, 15% EtOH and 400 mM KCl adjusted with phosphoric acid to pH 6.3 RT N2 - 100 mM Tris, 15% EtOH and 900 mM KCl adjusted with phosphoric acid to pH 6.3 RT N3 - 100 mM Tris, 15% EtOH and 1150 mM KCl adjusted with phosphoric acid to pH 6.3 RT N5 - 100 mM Tris, 15% EtOH and 1000 mM KCl adjusted with phosphoric acid to pH 8.5 RT
To make your own buffers or for more information please refer to Buffer Preparation and Nucleobond AX Buffers.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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