Method: Purification of Synthetic Oligonucleotides by Ion Exchange Chromatography

May 9, 1990

Terry C. Lairmore

Purpose:

This is a method for purifying synthetic oligonucleotides for use in PCR or sequencing reactions by ion exchange chromatography on a DEAE Sephadex column. While this purification step may be unnecessary for most purposes, it is relatively simple and quick and may be employed if there is concern about oligonucleotide purity. The resuspended stock solution of oligonucleotide in TE (after heating to 55 degrees C and drying in a rotary vacuum concentrator; refer to preparation of oligonucleotides in PCR Method) is purified over the columns as described below before spectrophotometric quantitation and the concentration adjusted to 50 µM.

Time required:

1-2 hours

Special reagents:

• DEAE Sephadex (Sigma A-50-120)
• Glass wool fiber

Procedure:

1. Make a DEAE Sephadex A-50 (Sigma) slurry of approximately 50% in TE (the DEAE Sephadex swells as a small amount is added to TE).
2. Pour an approximately 0.5 to 1.0 inch column of DEAE Sephadex in a small pasteur pipet with a small amount of glass wool fiber in the end. (Two sets of 5 ml snap-cap polypropylene round bottom tubes will be required for each sample. A hole large enough for the narrow end of the pasteur pipet column may be melted in the center of the cap of each tube using a red hot paper clip. The prepared column is inserted into the hole in the cap and the drains into the tube).
3. Wash the column several times with 1.0 ml of TE and discard the flow-through.
4. Load the oligonucleotide and cycle the 3 times, rinsing the original oligonucleotide tube with the last flow-through cycle (oligonucleotide binds to the column).
5. Wash the column with 2-3 ml of 150 mM NaCl/ 50 mM Tris (pH 8.0)/ 10 mM EDTA into the first set of tubes with liquid from step 4.
6. Elute oligonucleotide from the column into a clean tube using 1 ml of 1 M NaCl/ 50 mM Tris (pH 8.0)/ 10 mM EDTA and cycle the flow-through 3 times.
7. Wash the column one last time with 250 µl of 1 M NaCl/ 50 mM Tris/ 10 mM EDTA.

   (Retain columns and all eluted fluids until DNA is quantitated in final step.)

8. Divide oligonucleotide equally into 4 eppendorf tubes (each about 300-350 µl).

9. Add 1.0 ml cold absolute EtOH to each tube and chill at 4 degrees C for 1 hour or longer.
10. Spin for 15 minutes in a cold (4 degrees C) microcentrifuge and dry for 1-2 minutes in the rotary vacuum concentrator.
11. Resuspend each sample in a total of 500 µl TE or water. Dilute 5 µl of final product into 1.0 ml of TE or water (dilution 1:200) and read the absorbance at 260 nm (UV) in spectrophotometer. An absorbance at 260 of 1.0 equals approximately 33-35 µg/ml for oligonucleotides.

method after E. Green (personal communication)