Ethanol and Isopropanol/NaCl Precipitation for DNA-Encoded Library Intermediates

Why not evaporate or lyophilize?

Evaporation and lyophilization are the standard ways to remove HPLC eluent from a purified oligonucleotide, and they’re well described in the literature. In our hands, though, evaporating triethylammonium acetate (TEAA)/acetonitrile fractions caused massive degradation of many building blocks, and the resulting vials had a strong residual smell of ammonia — a sign that the triethylammonium counterion itself wasn’t just evaporating cleanly. That’s consistent with published reports of triethylammonium cation loss during the purification of nucleotide analogues more generally. Salt/alcohol precipitation sidesteps the problem entirely: the oligonucleotide-BB conjugate is pulled out of solution as a solid, while the TEAA, unreacted small molecules, and organic solvent stay in the supernatant.

The core protocol

The same basic recipe recurs at every stage of the process — after ligation, after conjugation, after HPLC pooling, and after deprotection — with only the volumes changing:

1. Add 4 M NaCl (roughly 1/10th to 1/15th of the well volume) to the aqueous sample.
2. Add ethanol (for single-well, smaller-volume precipitations) or isopropanol (for larger pooled volumes), typically at 2.5–3× the total aqueous volume for ethanol and 1x the total aqueous volume for isopropanol, and mix.
3. Incubate overnight at −20 °C.
4. Centrifuge 30 minutes at 4000 rpm, 4 °C.
5. Invert the plate to decant the supernatant, then spin briefly inverted (15 sec, ~200 rpm) to clear residual liquid.
6. Air-dry the pellet for about 1 hour.
7. Redissolve — typically overnight with agitation, to ensure complete dissolution of the oligonucleotide.

Volumes by stage

Why doing it in plates (not vials) matters

Precipitating pooled HPLC fractions directly in 48-well plates, rather than transferring each sample to an individual vial first, significantly reduces hands-on time. It also keeps the workflow contained: the same deep-well plate carries a sample from precipitation through redissolution, filtration, and back into the next reaction step, with liquid transfers kept to the two points where they’re unavoidable — filtering before HPLC, and collecting fractions afterward.

Practical notes

• Redissolution needs time, not just volume. Pellets were routinely given overnight agitation to fully redissolve — checking too early can look like incomplete recovery when it’s really just slow dissolution.
• A glass-fiber filter plate (1.0 µm) removes particulates before HPLC. Crude conjugate solutions were filtered through a 96-well glass-fiber filter plate after the first redissolution and before injection.
• This step is where TEAA gets removed. The HPLC purification runs in TEAA/MeCN, so the precipitation after fraction pooling is what actually clears the eluent — treat it as a required step, not an optional cleanup.