GenScript oligonucleotides produced with state-of-the-art oligo-synthesizers are of high purity with minimum truncated sequences and salt residuals.
However, improperly synthesized oligonucleotides from incomplete synthesis cycles and protecting groups cleaved from the bases after synthesis may exist in the final oligonucleotide samples in addition to the full length products. There are different methods for removing these impurities and key considerations in selecting the proper purification approach include the evaluation of the probable degree of contamination and the possible effects of these contaminants for downstream applications and experiments.
Purification with reverse-phase silica gel column which can effectively remove salts. The purity can meet the needs of most molecular biology experiments.
PCR amplification, gene synthesis, DNA sequencing, NGS capture probes, NGS Multiplex PCR oligos
Using denaturing polyacrylamide gel electrophoresis, oligo of different molecular weights migrate at different speeds during electrophoresis, allowing for separation. Finally, the target DNA is recovered from the gel.
Mutation libraries, protein binding gel migration electrophoresis analysis, modified oligos such as phosphate and biotin
The purification of oligo with HPLC can achieve high purity and is mainly used for short chain (< 40 nt) and modified oligo.
Point mutation, subcloning, diagnostic PCR, protein binding gel migration electrophoresis analysis, modified primers with hydrophobic groups.
PAGE purification done in a clean room.The target sequences are separated by denaturing polyacrylamide gel electrophoresis, and each oligo is purified with an independent purification system and disposable consumables to avoid the possibility of mixing with other oligos.
HPLC purification done in a clean room. Before purification of each oligo, columns are thoroughly cleared of residues by multiple special rinsing procedures, and different oligos are kept reasonably spaced apart and purified separately to avoid exogenous contamination.
| Purification Methods | Desalt | PAGE | HPLC | PAGE+ | HPLC+ |
|---|---|---|---|---|---|
| NGS adapters | Recommended | ||||
| NGS capture probes | Recommended | ||||
| NNGS multiplex PCR oligo | Recommended | ||||
| TaqMan probes, qPCR oligos | Recommended | ||||
| molecular biology experiments (PCR Primer/sequencing …) | Recommended | ||||
| <10 nt | Suitable | Not suitable | Suitable | Not suitable | Suitable |
| 10-39 nt | Recommended | Suitable | Recommended | Suitable | Recommended |
| 40-59 nt | Suitable | Suitable | Suitable | Suitable | Suitable |
| 60~120 nt | Suitable | Recommended | Suitable | Recommended | Suitable |
| Modifications | dI, dU, C3/6/9/12/18 etc. | dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA etc. | dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA CY5, BHQ, VIC etc. | dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA etc. | dI, dU, C3/6/9/12/18 P, S, dI, dU, LNA CY5, BHQ, VIC etc. |
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