The following protocol is a modified version of one that was successfully applied by Dr. Allan Pollock from the Nephrology Dept. of UCSF for the recovery of restriction fragments from low melting agarose gels. The purification was done on a 700 ng scale with nearly quantitative recoveries, using NUCLEOBOND AX 20 cartridges. This type of cartridge is suited for the isolation of up to 20 ”g of a particular restriction fragment.
1. Use one of the appropriate buffer systems for the electrophoretic separation of DNA fragments.
2. Cut out the electrophoretic band and place in a buffer of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 200 mM KCl and add an equal volume of phenol to the solution.
3. Freeze and thaw 3x on dry ice.
4. Centrifuge and collect the aqueous supernatant very carefully. Add 2 volumes of buffer N2. (For fragments smaller than 100 bp in size, substitute buffer N1 for N2 in this step.) Check that the pH of the final solution is 6.3. If it is not, adjust as required with dilute phosphoric acid
5. Equilibrate the NUCLEOBOND AX 20 cartridge with 1 ml of buffer N2. (As in step 4, for fragments smaller than 100 bp in size, substitute buffer N1 for N2 in this step.)
6. Load the diluted aqueous supernatant of step 4 into the cartridge.
7. Wash the cartridge twice with 1 ml of buffer N3. (For fragments smaller than 100 bp, substitute N2 for N3 in this step.)
8. Elute dsDNA fragment with 0.9 ml buffer N5.
9. Precipitate the DNA by adding 0.8 volumes of iso-propanol and centrifuge at 12,000 x g for 10-15 minutes.
10. 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.
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.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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