Desalting Protocol

Our unpurified product contains various organic salts, including benzamide, isobutyramide, and ammonium acetate which are utilized during synthesis to protect both the backbone and the bases. The following procedure utilizes a disposable reverse-phase cartridge to remove these salts.

ULTRAMILD OLIGOS: Before desalting UltraMild oligos that have been cleaved and deprotected using potassium carbonate in MeOH, the MeOH content must be reduced to < 5% through speed-vac'ing or dilution with "A" Buffer. An organic content > 5% will prevent DNA from binding to the Sep-Pak resin! Adding TE to UltraMild oligos is not required.

The DNA Glen-Pak cartridges can be used for desalting DNA or RNA oligonucleotides directly after deprotection or post purification by HPLC and Polyacrylamide Gel Electrophoresis (PAGE).    The cartridges are designed specifically for DMT-ON purification where failure sequences not containing a 5’ DMT are eluted with salt washes, but when an oligo is loaded in 0.1M TEAA instead of 100mM sodium chloride, a DMT-Off oligo may also be captured on the column. As with other SPE methods, it is suggested that the oligo be applied to the cartridge in an aqueous solution or one containing less than 5% organic solvent. It is always prudent to keep loading and rinse volumes until the purified product is quantified. Of special note is that the DMT-Off method for crude, deprotected oligos below uses the Glen-Pak DNA cartridge for desalting of BOTH RNA and DNA oligonucleotides. This will allow our customers currently using more than one cartridge platform for downstream processing to harmonize to only one column type for DMT-Off desalting.

Materials
Glen-Pak DNA Purification Cartridge (60-5100-XX, 60-5200-XX)
Vacuum manifold (96 well or 12-24 port SPE type if appropriate)
HPLC Grade Acetonitrile
2.0M Triethylamine Acetate (60-4110-XX, TEAA, pH7)
0.1M TEAA, pH7 Deionized Water (RNase Free for use with RNA oligonucleotides)
50% Acetonitrile/Water containing 0.5% ammonium hydroxide*
10% Acetonitrile/Water (for desalting of crude RNA and DNA)

* Add 50uL of concentrated Ammonium Hydroxide per 10 mL of 50% Acetonitrile solution.

Sample Preparation
1.    HPLC purified oligos: Add the required amount of 0.1M TEAA to the post purification sample to obtain ≤ 5% final concentration of acetonitrile.
2.    PAGE purified oligos: Elute purified oligonucleotide from the gel slices in 0.05M TEAA as described in current protocols, filter away gel slices and bring up to at least 2 mL in 0.1M TEAA.
3.    DMT-Off, crude, deprotected oligos in NH4OH or AMA: Evaporate the deprotected oligo to dryness and reconstitute in 2 mL 0.1M TEAA.

Cartridge Preparation
4.    Prepare the cartridge as described for DMT-ON purification.

Desalting Procedure HPLC Purified Oligos:
5.    Load the solution containing the oligonucleotide onto the cartridge.
6.    Flush the cartridge with 2 x 1 mL of deionized water. This wash removes the salts from
the cartridge.
7.    Elute the desalted oligonucleotide by flushing the cartridge with 1 mL 50% Acetonitrile/water containing 0.5% ammonium hydroxide. The product should elute fully in 1 mL of 50% Acetonitrile with 0.5% ammonium hydroxide.

Desalting Procedure PAGE Purified Oligos:
8.    Load the solution containing the oligonucleotide onto the cartridge.
9.    Flush the cartridge with 2 x 1 mL of deionized water. This wash removes the salts from
the cartridge.
10.    Elute the desalted oligonucleotide by flushing the cartridge with 1 mL 50% Acetonitrile/water containing 0.5% ammonium hydroxide. The product should elute fully in 1 mL of 50% Acetonitrile with 0.5% ammonium hydroxide.

Desalting Procedure Crude Deprotected DNA Oligos:
11.   Load the solution containing the oligonucleotide onto the cartridge.
12.   Flush the cartridge with 2 x 1 mL of 0.1M TEAA.
13.   Flush the cartridge with 2 x 1 mL deionized water. This wash removes the salts from the cartridge.
14.   Elute the desalted oligonucleotide by flushing the cartridge with 1 mL 10% Acetonitrile/water. The product should elute fully in 1mL while leaving behind organics such as benzamide on the column matrix.

Desalting Procedure Crude Deprotected RNA Oligos:
15.   Conduct 2’ deprotection and quenching of DMT-Off RNA oligonucleotides as suggested in the technical bulletins for TBDMS and TOM RNA monomers.
16.   Load the resultant 2 mL of deprotected/quenched RNA solution directly on a Glen-Pak DNA cartridge that has been prepared using the same methods as above.
17.   Rinse with 2.0 mL 0.1M TEAA (Fresh 2.0M TEAA diluted in RNase free water)
18.   Rinse with 2.0 mL RNase free water.
19.   Elute the desalted product in 10% Acetonitrile in RNase free water.

Oligonucleotide Storage:
20.    Note: Acidic conditions can damage oligonucleotides so it is important to keep the oligonucleotide solution basic (pH > 7.5) during either drying or storage of the eluted product. It is always safer to store oligos in a buffered solution such as 10mM Tris-HCl, 1mM EDTA, pH 8.0 (TE Buffer).
21.    Determine the yield and store purified oligonucleotide lyophilized solid at -20°C.
Glen-Pak desalting should yield over 90% recovery of the original product.

Reposted from Glen Research GlenPak _User Guide.