Procedure for the purification of double-stranded DNA (>100 bp) from reactions

The following protocol was developed for the isolation of DNA of 100 bp or longer from reaction mixtures containing enzymes, nucleotides, or other reagents. The choice of cartridge to use, Nucleobond AX-5 or AX-20, will depend on the amount of DNA to be purified.

Special conditions for the type of NUCLEOBOND AX cartridge used

Step of the procedure AX 5 AX 20 Maximum amount of dsDNA 5 g 20 g

1. Equilibrate the cartridge with 0.5 ml 1.0 ml buffer N2.

2. Add at least 4 volumes of buffer yes yes N2 to the reaction, mix carefully, and check that the pH is no higher than 6.3. If it is, titrate it as required with dilute phosphoric acid.

3. Apply the solution to the cartridge. yes yes 4. Wash the cartridge with buffer N3 2 x 1.0 ml 2 x 2.0 ml to remove contaminants.

5. Elute the DNA with buffer N5. 2 x 0.4 ml 2 x 0.6 ml

6. Add 0.8 volumes of iso-propanol to 0.5 ml 1.0 ml the eluate. Precipitate for 20 minutes at 4C and centrifuge for 10-15 minutes at 0C at 15,000 x g.

7. Wash the pellet with 70% ethanol. The yes yes pellet should be air dried briefly and dissolved in an appropriate buffer for further manipulations.

Buffer solutions                                          Storage

N2 - 100 mM Tris, 15% EtOH and 900 mM KCl adjusted RT with phosphoric acid to pH 6.3 N3 - 100 mM Tris, 15% EtOH and 1150 mM KCl adjusted RT with phosphoric acid to pH 6.3 N5 - 100 mM Tris, 15% EtOH and 1000 mM KCl adjusted RT with phosphoric acid to pH 8.5

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.

Back to the Nucleobond AX applications guide

Updated 1/20/98