Desalting Oligonucleotides by Ethanol Precipitation

1. Measure the volume of the oligonucleotide solution to be precipitated.
2. Chill 95% Ethanol in the freezer at -20 °C or on dry ice prior to use.
3. Perform Calculations:

   Volume of 3 Molar Sodium Acetate pH 5.2 = 0.1 * Volume of Oligonucleotide Solution

   Volume of 95% Ethanol = 2.5 * (Volume of Oligonucleotide Solution + Volume of 3 Molar Sodium Acetate pH 5.2)

4. Add the calculated volume of 3 Molar Sodium Acetate to the oligonucleotide solution and vortex for 10 seconds to mix.
5. Add the calculated volume of 95% Ethanol to the oligonucleotide solution and vortex for 10 seconds to mix.
6. Place the reaction mixture in the freezer at -20 °C or on dry ice for at least 1 hour.
7. Centrifuge the reaction mixture to pellet the precipitated oligonucleotide. A small, gray pellet should now be visible in the bottom of the tube.
8. Pipette off the supernatant and discard.
9. Add the same volume of cold 95% Ethanol to the pellet and vortex. It is important to break up the pellet to sufficiently wash out trapped salts.
10. Repeat Step 7 through 9 once more.