Purification Methods for Oligonucleotides
Why Purify Oligos?
When oligonucleotides are first made, the reaction also creates by-products. These can include short, incomplete strands or fragments without the intended modifications. Purification removes these extra products and enriches the full-length sequence, which improves performance in downstream applications like PCR, qPCR, sequencing, or cell-based studies.
Common Purification Options
| Method | Best For | Typical Outcome |
|---|---|---|
| Desalting | Oligos ≤35 bases | Removes small molecules and salts. Good enough for most PCR or short primers. |
| Ethanol Precipitation | Crude oligos or buffer exchange | Simple way to clean up and concentrate an oligo before use. |
| Cartridge Purification | Oligos with a dye or 5′-modification | Separates longer full-length strands from shorter by-products. |
| Reverse-Phase HPLC (RP-HPLC) | Fluorescent probes or medium-length oligos | Provides higher purity; efficient for labeled oligos. |
| Ion-Exchange HPLC (IE-HPLC) | Oligos with secondary structure or high GC content | Separates based on charge; helpful when standard methods struggle. |
| Polyacrylamide Gel (PAGE) | Long sequences (≥50 bases) or when the highest purity is required | Achieves very high purity but with lower overall yield. |
| Gel Filtration | Oligos for in vivo work | Removes trace impurities that could cause cell toxicity. |
| Dual Purification | Demanding or regulated applications | Combines two methods for maximum purity. |
Quick Guide: Choosing a Method
-
Primers for PCR (short): Desalting is usually sufficient.
-
Probes or dye-labeled oligos: Cartridge or RP-HPLC is recommended.
-
Sequences with lots of GC or structure: IE-HPLC works best.
-
Very long oligos or applications needing the cleanest product: PAGE purification.
-
Therapeutic or in vivo use: Gel filtration or dual purification for extra safety.
Takeaway
Not every oligo needs heavy purification. For many routine uses, simple desalting works perfectly well. But for specialized probes, longer sequences, or sensitive experiments, advanced methods like HPLC or PAGE give the confidence of higher purity.
If you’re unsure which purification level is right for your application, our team can recommend the best option based on sequence length and intended use.