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

Special conditions for the type of NUCLEOBOND AX cartridge used
Step of the procedure                     AX 5              AX 20
Maximum amount of dsDNA                       5 µg              20 µg

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