Purifications
Each oligonucleotide deserves 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 choosing the most appropriate purification method for your oligo you must take into account various parameters such as the oligo length, the presence of modifications and your application.
We offer purifications reaching up to 95% purity level
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 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 provides a degree of purity of at least 85%. Reverse Phase (RP) is based on the hydrophobic interaction of the full-length oligonucleotides with alkyl chains bonded on the matrix. Ion exchange (IEX) is based on the preference of the anion-exchange resins (positively charged) for the full-length oligonucleotides.
In vivo oligonucleotides are the smart choice for antisense oligonucleotides or siRNA testing at a research level before entering into preclinical studies. The production process of in vivo oligonucleotides includes the following steps: IEX-HPLC purification, desalting, filtration and lyophilization.
Polyacrylamide Gel Electrophoresis (PAGE) separates oligonucleotides varying from only 1 base and gives a purity level of 85-90%. Gel band is excised under low intensity UV. Oligonucleotide is then eluted, precipitated, quantified and packaged.
Dual HPLC (double RP or RP+IEX) increases the purity level up to 95%
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 |
| 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 |