DMT-On Purification Protocol using Glen-Pak Cartridges

Glen-Pak Purification Cartridge

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)
100 mg/mL Sodium Chloride
Salt Wash Solution (5% Acetonitrile in 100mg/mL Sodium Chloride)
2% Trifluoroacetic Acid (TFA)/Water (60-4040-57)
Deionized Water
50% Acetonitrile/Water containing 0.5% ammonium hydroxide*

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

Sample Preparation
1.    Following DNA synthesis, deprotect the DMT-ON oligonucleotide in 1mL of ammonium hydroxide or ammonium hydroxide/methylamine (AMA) as normal. There is no need to lyophilize the deprotection solution. For this procedure to be successful, the synthesis must have been carried out DMT-ON. The base labile protecting groups must have been removed with either AMA or ammonium hydroxide (1.0mL).
2.    Add 1 mL of 100 mg/mL Sodium Chloride solution to the deprotected DMT-ON oligonucleotide for a final volume of 2 mL. The final salt concentration of the sample must be around 50 mg/mL for the loading of the oligo on the cartridge. Larger volumes may be loaded, but this is our suggested volume for loading on the cartridge.
Cartridge Preparation
3.    Place the desired number of 150 mg cartridges into the female luer ports of the manifold and collection tubes (if desired) in the rack below the output guides. We routinely use a 12-port manifold.
4.    Turn on the vacuum and adjust the pressure to ~7 mm Hg using the vacuum control valve. (If no control valve is available on your manifold, target a flow rate of about 1-2 drops per second). Condition the cartridge using 0.5mL of Acetonitrile followed by 1 mL 2M TEAA. The acetonitrile washes organic residues from the resin and wets it, while the TEAA acts as an ion-pairing reagent to enhance the binding of the DMT-ON oligonucleotide to the resin.
Purification Procedure
5.    Apply the oligo/salt mixture to the cartridge in 1 mL aliquots (collect the eluent and save in case of loading failure or error). During the loading process, the DMT-ON oligos tend to stick to the cartridge packing material while most of the failure sequences are not retained.
6.    Wash the cartridge with 2 x 1 mL of Salt Wash Solution. This rinses away the remainder of the failure sequences from the cartridge.
7.    Rinse the cartridge with 2 x 1 mL of 2% TFA. A faint orange band may be visible while the DMT is removed from the bound, full-length oligonucleotide.
8.    Wash the cartridge with 2 x 1 mL of deionized water. This rinses away the TFA and excess salts.
9.    Place the appropriate receptacle (96 deep-well plate or sample tube) into the manifold and elute the purified oligo using 1 x 1 mL 50% acetonitrile in water containing 0.5% ammonium hydroxide. If desired a second elution can be performed to ensure full recovery, but in our experience, full recovery of the oligonucleotide is achieved in this one step.
10.    Note: The dilute ammonium hydroxide is utilized to neutralize any remaining TFA. 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).
11.    Determine the yield and store purified oligonucleotide lyophilized solid at -20°C.
Glen-Pak DNA purification should yield over 90% recovery of the original DMT-ON product. The actual yield depends on synthesis efficiency and the amount of failures in the oligonucleotide sample.

 

Reposted from Glen Research GlenPak _User Guide.