Oligonucleotide FAQ

How are oligonucleotides synthesized?

Oligonucleotides are synthesized using phosphoramidite chemistry. Details of our synthesis methods can be found in our DNA Oligonucleotide Synthesis article.

What is the longest sequence that I can order?

Standard DNA Oligos are available up to 120 bases. However, Long Oligos—specifications are more limited compared to Standard DNA Oligos—are available up to 180 bases. For details, please review our Long Oligos offering.

What is the shortest sequence that I can order?

Standard DNA Oligos are available as short as 6 bases. If you need less than 6 bases, please contact dnaoligos@sial.com for feasibility assessment / quotation.

Does my oligonucleotide have a phosphate on the 5' or 3' end?

All oligonucleotides are synthesized with a hydroxyl group on each end. However, phosphate can be added to both ends. Please add this modification to your sequence in either the online ordering configurator or the email ordering template. The code for phosphate is [Phos] and looks like this in the sequence:

[Phos]ACGTACGTACGTACGT[Phos]

What modifications are available?

Standard modifications available by category (fluorescent, attachment, binding, spacer, analog, intercalation, antisense, and phosphorothioate) can be found on our Custom DNA Oligos Modifications page. If you do not see your required modification, there is a good chance that we can still work with it, especially if it is commercially available as a CPG, phosphoramidite, or NHS ester.

If not commercially available, there is a chance that we can manufacture your modification as a raw material (for feasibility, please email dnaoligos@sial.com with a description of your needs; a minimum spend of $25,000 and a lead time of six months often applies).

When designing my sequence, where should I place modifications?

This depends on the requirements of the intended technique and application. For example, with a qPCR probe, a 5' reporter dye and a 3' quencher is considered standard, is most commonly requested, and works well. However, some users may require an internal quencher for a variety reasons, which we might be able to manufacture upon request. For qPCR probes that require shorter sequence lengths due to targets with AT-rich regions, LNA^® (Locked Nucleic Acid^®) can be added internally. For assistance with modification placement, please contact our technical services team at oligotechserv@sial.com.

Do you offer alternative dyes to those that are protected by intellectual property?

Yes, if you intend to commercialize your oligonucleotides, it is understandable that you want dyes giving you freedom to operate. Please contact our technical services team at oligotechserv@sial.com, and we will determine what alternatives are available for the dyes you want to replace.

What purification methods are available and what are their guaranteed purities?

Our methods and guaranteed purities, include: desalt (no guarantee), cartridge (no guarantee, but 65-80% full-length sequence is typical), RP-HPLC (Reverse-Phase High Performance Liquid Chromatography, >85% full-length sequence), and PAGE (Polyacrylamide Gel Electrophoresis, >95% full-length sequence). While less-commonly requested, we also offer IE-HPLC (Ion-[specifically, Anion]-Exchange HPLC, please inquire for the guaranteed full-length sequence). Finally, we also offer NGSO (Next-Gen Sequencing Oligos), which are custom adapters purified with proprietary processes, the purposes of which are to drive down cross contamination to the lowest levels possible. Details of our purification methods can be found in our Oligonucleotide Purification article.

Why do the analytical HPLC chromatograms of phosphorothioate oligonucleotides appear broad?

The presence of sulfur creates a stereogenic α-phosphorus, and the resulting diastereomers have subtly shifted elution times during chromatography. Details of the problem can be found in our Chromatography Profile Analysis of Phosphorothioate Oligonucleotides article.

How do you determine the quantity?

We use the following method to determine oligo quantity: 1) measure A₂₆₀ values using a UV-Vis spectrophotometer; 2) convert the absorbance value to concentration using the Beer-Lambert Law; 3) convert the concentration to customary units of optical density (OD). Details and example calculations of the method can be found in our Oligonucleotide Quantification article.

If I order the 0.2 µmol scale, will I receive 0.2 µmol of material?

No, because the amount of material delivered is always less than the starting synthesis scale (you can request an exact amount of material as opposed to ordering by starting synthesis scale). This is because the coupling efficiency between each base is <100%. In addition, cleavage, deprotection, and purification decrease the yield further (see the section titled, ‘Yield’ at the bottom our DNA Oligonucleotide Synthesis article for additional details). For minimum yield guarantees, please review our Standard DNA Oligos offering.

Why do I not see any material in the vessel (tube / plate well)?

Unless specified differently, all oligonucleotides are provided dry. If you hold the tube / plate in light at the right angle, you might see the material as a thin film or glaze at the bottom of the vessel. Before removing caps / plate mats, we recommend centrifugation just in case the material came loose during transit. Resuspension in water or buffer and an A₂₆₀ reading should provide confidence that the oligonucleotide is present.