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Primers and Oligonucleotides – General Information and References

Purification Options

Standard purification
Standard purified oligos are deprotected and desalted, recommended for high through-put methods like PCR, (cycle) sequencing and hybridisation without any restrictions.

OPC purification (Oligonucleotide Purification Cartridge™)
OPC purification is a kind of fast HPLC purification for unmodified oligos up to about 40 nucleotides in length. Non-DMT bearing failure sequences, by-products, and other impurities wash through the cartridge while the DMT group of the trityl-on oligos adsorb to the affinity column. After removal of the DMT group with mild acid the highly purified detritylated primer can be eluted.

HPLC purification
HPLC purification by a computer controlled HPLC-system is the state of the art method to precisely enrich for long full-length products. Its sensitive separation capability makes it possible to get purities up to 95%. Our special developed HPLC programs aren't "2-minute one cleans all kind of oligos programs" as you'll find it by many of our competitors. We have different programs for different types of oligos which have a run time up to 30 minutes. As a documentation of your oligos quality you'll get the HPLC chromatogram with your primers.
We strongly recommend HPLC purification for all modified oligos!

Very long oligonucleotides
From our experience in nucleotide synthesis chemistry you should avoid to design oligo-/polynucleotides longer than 80 bases for high precise applications. Even if we can do up to 180-mers, the yield of full-length product will (coupling rates/cycle ~ 98.5-99.5%) dramatically decrease with the number of synthesis cycles and (de) protection, coupling and washing steps within each cycle can leave its track within the growing sequence (base modifications by oxidation, base insertions/deletions by formation of secondary structures, ...). Consequently, a precise sequence guarantee is very difficult to give and we have to double the price per base for oligos >80 nucleotides.

Quality Control and Documentation

Quality control is the last step in the production process of custom oligonucleotides. Our Quality Management starts before the first step!

  • Pre-synthesis control: Validation of incoming raw material/chemicals by running and checking test oligos makes sure that our reagents, which are exclusively purchased from audited and approved quality vendors, meet the requirements for providing best coupling efficiencies and consequently top quality primers.
  • In-synthesis control: During synthesis coupling rates are permanently checked by DMT monitoring - only high efficient (>98.5 %) cycles allow the continuation of the synthesis run!
  • Post-synthesis control: Quantification (OD measurement) of the end product is a strong indicator for successful synthesis.
  • End control: Analytical HPLC and Maldi-ToF spot checks are performed for final release of the product.
Our strict maintenance program for regular testing of the performance of our synthesizers is an additional "quality measure" taken from our side to guarantee our constantly high quality level.

Handling and Storage of Oligonucleotides

Oligonucleotides are biomolecules with a limited shelf life!

To gain a maximum shelf life for oligonucleotides, samples should generally be stored dehydrated at ≤-15°C in absence of light. Under the mentioned conditions, samples are stable for at least 12 months. In case of a longer storage period, oligos should be pretested for molecular integrity prior to experimental use.

The shelf life of oligonucleotides is determined by three main parameters:

1. pH sensitivity
Single strand oligos are sensitive to acidic pH values. Therefore the solution media for solving the lyophilised oligonucleotide should be neutral or of low alkalinity (pH 7-9).

2. Nuclease degradation
Oligonucleotides can be catabolized by nuclease activity. Nuclease motivated degradation of oligonucleotides is accelerated in the presence of bivalent and/or trivalent cations. To minimize contamination with nucleases, preventive hygiene measures should be taken (wearing laboratory gloves while handling oligos etc.).

3. Freeze-thaw effects
The integrity of oligonucleotides can be affected by freeze-thaw processes. To avoid any negative influence on the oligo quality, these procedures should be limited to a minimum. Therefore, we recommend to aliquot the originally delivered stock solution. In case oligonucleotides are delivered as dry material, reconstitute the product in purified water, TE or any biological buffers suitable for your application at a concentration of no less than 100 μM (pH of 7-9, see point 1 above), and produce aliquots. Avoid the use of distilled water, since solution pH may be as low as 4-5.

Recommended further procedure and storage conditions for modified oligonucleotides (especially fluorescent dye labelled oligos): Don’t re-lyophilise but store highly concentrated aliquots dark and frozen at ≤ -15°C, thaw them only once, produce working dilutions just before usage and keep them dark at 4°C. We recommend using up working dilutions within 4 days from the date of preparation. Any unused leftover should be discarded.

Please note:

  • Minimize material’s exposure to light because of its bleaching effect.
  • The higher the dilution factor, the faster fluorescent activity fades away.
  • Diluted working solutions shall be stored refrigerated, not frozen.

General information on DNA oligonucleotide delivery & documentation

If not ordered otherwise, all custom oligo products are delivered without a 5’ (or 3’) phosphate group, fully deprotected and ready-to-use. Only Thiol-modified oligonucleotides are supplied in the protected form with the disulfide linkage intact to minimize the potential for oxidation, which results in oligo dimer formation. To use the free thiol (– SH) in your application the disulfide linkage must be reduced.

Appearance of dry oligos may vary from powdery to hyaloid, transparent to off-white/yellowish for unmodified oligos, and respectively colored for dye-labeled oligos. Depending on the amount and density of dried DNA oligonucleotide, solubility and appearance may differ.

Oligos are quantified by measuring their absorbance of ultraviolet light at 260 nm in neutral aqueous solution (Optical Density/OD measurement). Quantity delivered as well as other relevant physical oligo properties are recorded on a comprehensive “Synthesis Report” (Certificate of Analysis) coming along with the products. If HPLC purification or a final QC check by MALDI or ESI-ToF has been ordered, respective chromatograms/Mass spectra are additionally enclosed.

Deprotection of Thiol-modified Oligonucleotides - short protocol

Just prior to use, cleave the disulfide with 100 mM DTT (pH 8.3-8.5) at RT for about 30 minutes. Thiol-modified deprotected oligonucleotides should be kept either under an inert atmosphere or in a solution containing DTT (10 mM) to avoid disulfide formation.