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Oligonucleotides
Purifications
Choose the right purification method
The aim of any purification method is to remove the by-products resulting
from the removal of the protecting groups and other synthesis by products.
When chosing the most appropriated purification method for your oligo you must take into
account various parameters such as the oligo length, the presence of modifications and your application.
SEPOP DESALTING
SePOP desalting increase the purity level of the deprotected and desalted oligos up to 65-70%. It uses differential precipitation to eliminate the largest part of contaminants (truncated material < 10 bases).
RP Cartridge•Gold
RP Cartridge•Gold consists of a reverse phase chromatography based on the difference in hydrophobicity between the full-length product and truncated sequences. It yields to 75-80% purity. It is the best compromise for most applications and the absence of residues (which may occur with HPLC) makes them suitable for cell culture uses.
HPLC
HPLC will give you at least 85% up to 45 bases (standard sequences). Not applicable for double-dye and MGB probes (80% min purity) or double modified oligos. A lower purity may result from poly-modifications and/or strong secondary structures.
PAGE
Page will give you at least 85% of purity up to 79 bases (standard sequences). Not applicable for double modified oligos. A lower purity may result from poly-modifications and/or strong secondary structures.
Dual HPLC
Dual HPLC will give you a purity of at least 85% purity for ≤45 bases oligos. Appropriate for antisense oligos or siRNAs, might be required for probes with post-synthesis coupling.
In Vivo like
In vivo like purification will give you at least 85% up to 39 bases (standard sequences). Not applicable for double modified oligos.
A lower purity may result from poly-modifications and/or strong secondary structures.
In vivo like purification process includes:
- Ion exchange HPLC
- Desalting
- 0,2 μm filtration
- Lyophilization
Applications vs Purifications
| Applications (by alphabetical order) | Recommended Purification |
|---|---|
| AFLP | RP Cartridge•Gold |
| Capillary sequencing | HPLC |
| Cloning and subcloning PCR | Polyacrylamide Gel Electrophoresis (PAGE) |
| Cycle sequencing | SePOP desalting |
| DNA MicroArray | SePOP desalting |
| First-strand cDNA synthesis | Polyacrylamide Gel Electrophoresis (PAGE) |
| Gel-shift assay | Polyacrylamide Gel Electrophoresis (PAGE) |
| Gene synthesis | Polyacrylamide Gel Electrophoresis (PAGE) |
| Hybridization | SePOP desalting |
| in situ Hybridization | HPLC |
| in vivo studies | In vivo |
| Isothermal sequencing | SePOP desalting |
| miRNA, siRNA and antisense | HPLC |
| NGS | HPLC |
| NMR | UltraPureGold™ |
| OLA | RP Cartridge•Gold |
| PCR primers | RP Cartridge•Gold |
| Production of cloning linkers | Dual HPLC |
| Real-Time qPCR | HPLC |
| Routine PCR | SePOP desalting |
| Sensitive PCR (Diagnostic) | RP Cartridge•Gold |
| Site-directed mutagenesis | Dual HPLC |
| SNP Analysis | SePOP desalting |
| X-ray crystallography | UltraPureGold™ |