Procedure for the purification of RNA from reactions

The following protocol was developed for the isolation of RNA from reaction mixtures containing enzymes, nucleotides, or other reagents. The choice of cartridge to use will depend on the amount of RNA to be purified. Please read the entire protocol including footnotes before attempting.

Special conditions for the type of NUCLEOBOND AX cartridge used

Step of the procedure AX 5 AX 20 AX 100 Maximum amount of RNA 20 g 80 g 400 g

1. Equilibrate the cartridge with 0.5 ml 1.0 ml 2.0 ml buffer N1. (NOTE 1)

2. Add at least 4 volumes of buffer yes yes yes N1 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. (NOTE 2)

3. Apply the solution to the yes yes yes equilibrated cartridge.

4. Wash the cartridge with buffer 2 x 1.0 ml 2 x 2.0 ml 2 x 4.0 ml 1:1 N1/N2 to remove contaminants. (NOTE 1)

5. Elute the RNA with buffer N5. 2 x 0.4 ml 2 x 0.6 ml 2 x 1.4 ml (NOTE 3)

6. Add 0.8 volumes of iso-propanol 0.5 ml 1.0 ml 2.2 ml to 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. yes yes yes The pellet should be air dried briefly and dissolved in an appropriate buffer for further manipulations.

Buffer solutions Storage N1 - 100 mM Tris, 15% EtOH and 400 mM KCl adjusted with RT phosphoric acid to pH 6.3 N2 - 100 mM Tris, 15% EtOH and 900 mM KCl adjusted with RT phosphoric acid to pH 6.3 N3 - 100 mM Tris, 15% EtOH and 1150 mM KCl adjusted with RT phosphoric acid to pH 6.3 N5 - 100 mM Tris, 15% EtOH and 1000 mM KCl adjusted with RT phosphoric acid to pH 8.5

To make your own buffers or for more information please refer to Buffer Preparation and Nucleobond AX Buffers.

NOTE 1: Please refer to Buffer Preparation for recommended N series buffer preparation procedure. Buffers comparable to N1 and 1:1 N1/N2 can also be made as follows: (1) for N1, substitute 50% N2 in 15% ethanol, pH 6.3 and make by mixing equal volumes of N2 and 15% ethanol, pH 6.3, for example, mix 5.0 ml of N2 and 5.0 ml of 15% ethanol, pH 6.3; (2) for 1:1 N1/N2, substitute 50% N2 from above for N1 and make by mixing equal volumes of N2 and 50% N2, for example, mix 5.0 ml of N2 and 5.0 ml of 50% N2.

NOTE 2: The ethanol, iso-propanol, and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the reagent approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 g of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.

NOTE 3: The N5 buffer will also elute any DNA that is left on the column after the washes. If the reaction contains DNA, such as in the production of cRNAs, and it is desired that the RNA be purified away from the DNA as well as the other reaction components, then elute the RNA with buffer N3 in step 5 and complete the procedure as usual from step 6.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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Updated 1/20/98