Prepare a Sep-Pak (C18) cartridge column and a 10 mL syringe. Ensure all components are clean and free of contaminants.
Fill the 10 mL syringe with Acetonitrile (ACN). Connect the syringe to the inlet of the cartridge. Gently push the plunger to wash the column with ACN at a flow rate of approximately 2 drops per second. Keep the column matrix wetted for the next step.
Using the same method, wash the column with 10 mL of deionized (DI) water to remove excess organic solvent.
Wash the column with 5 mL of 100 mM ammonium acetate solution. This prepares the column environment for optimal oligonucleotide binding.
Prepare your oligonucleotide sample in an aqueous solution (ensure organic solvents are minimized, volume < 5 mL). Draw the sample into the syringe, connect it to the cartridge, and load it very slowly at a rate of 1 drop per second.
Important: From this step forward, collect and save the flow-through solution. Do not discard it until the final purification and recovery are confirmed.
To remove salts and small impurities, wash the sample-loaded column with 10 mL of DI water. Repeat this wash step three times.
Draw 7 mL of 30% methanol (in water) into the syringe. Connect it to the cartridge and elute slowly. Collect the eluent in 1 mL fractions (using separate tubes). Measure the UV absorbance at 260 nm for each fraction to identify and pool the fractions containing the oligonucleotide.
Remove the solvent using a rotary evaporator, SpeedVac, or lyophilizer (freeze dryer). Once dried, redissolve the purified oligonucleotide in your desired volume of DI water to obtain the final product.