The following protocol was validated for the purification of single-stranded oligonucleotides from radiolabelled triphosphates and other molecular weight contamination by S. Arends and A. Bauer, Biozentrum, University of Wurzburg, FRG, using NUCLEOBOND AX cartridges. Oligonucleotides of about 20mer to 100mer can be purified with this procedure with recoveries of 90% or better. The separation of shorter oligos is achieved by reducing the salt concentration in the washing buffer to 150 mM KCl by dilution with 15% ethanol, pH 6.3.
Step of the procedure AX 5 AX 20 Maximum amount of oligonucleotide 5 µg 20 µg Special conditions for the type of NUCLEOBOND AX cartridge used
1. Equilibrate the Nucleobond AX 0.4 ml 1.0 ml cartridge with buffer U1
2. Add buffer U2 to the reaction vial, 1.0 ml 2.0 ml mix carefully and check that the pH is not greater than 6.3. If it is, titrate it as required and apply the solution to the cartridge.
3. Wash the Nucleobond AX cartridge with 2 x 1.0 ml 2 x 2.0 ml buffer U2 to remove low molecular weight contaminants.(e.g.[a 32P-ATP]).
4. Wash the Nucleobond AX cartridge with 1.0 ml 2.0 ml the buffer U0.
5. Elute the oligonucleotide with buffer U3. 2 x 0.5 ml 2 x 0.9 ml
6. Add 5 volumes of ethanol / acetone 5 ml 9 ml (1 vol. / 3 vol.) to the oligonucleotide solution. Precipitate at -20C for at least 70 minutes.
7. Centrifuge for 40-60 min. at 0C at yes yes 15,000 x g.
8. The supernatant is discarded and the dry pellet is stored at -20C or dissolved in an appropriate buffer.
Buffer solutions Storage
U0 - 100 mM Tris-Acetate, pH 6.3, 15% ethanol RT U1 - 100 mM Tris, 15% ethanol and 100 mM KCl adjusted RT with phosphoric acid to pH 6.3 U2 - 100 mM Tris, 15% ethanol and 200 mM KCl adjusted RT with phosphoric acid to pH 6.3 U3 - 600 mM Tris-Acetate, 15% ethanol, pH 8.5 RT
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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