Oligonucleotide synthesis services

We have successfully synthesized 220-nt unmodified DNA and 160-nt unmodified RNA. Due to their challenging nature, we evaluate the feasibility of such requests case-by-case and cannot guarantee the yield and purity of such long sequences.

We offer 5-75 µmol starting scales for most oligos on a regular basis. The minimum scale of 1 µmol is available if you order 5 sequences. For large amounts, the starting scale can be >75 µmol. Please visit trilinkbiotech.com/oligobuilder or contact [email protected] for more information.

Stability can differ with each individual sequence. To prolong their stability to >6-12 months, we recommend:

• Storing your oligos at -20 °C or lower
• Keeping them in a nuclease-free environment
• Minimizing freeze-thaw cycles.

We recommend: 

• Spinning down the tube to collect the oligo at the tube bottom.
• Calculating the required volume for your desired concentration.
• Using nuclease-free water or a desired buffer (e.g., Tris EDTA) at pH ~7-8 to resuspend

Hydroxyl (OH) groups are on the 5′ and 3′ ends of our unmodified DNA or RNA oligos.

We can provide oligos with modifications that are not listed on our website. Please submit your inquiry at trilinkbiotech.com/oligobuilder or contact us at [email protected] with details. We will assess and respond to your request.

Yes, please submit your request at trilinkbiotech.com/oligobuilder.

A phosphodiester linkage has oxygen on all phosphate bonds and is a common linkage between the nucleotides. A phosphorothioate linkage has a sulfur in a non-bridging location on the phosphate backbone. This modification is commonly incorporated to reduce nuclease degradation of oligonucleotides.

Since it is very challenging to distinguish between the two species, we do not offer isomer-specific phosphorothioate-modified oligonucleotides at this time.

We recommend no more than one conjugate every 5-10 bases, including those on the 5’ and 3’ ends. A higher number of conjugation can impact synthesis efficiency, labeling analysis, oligo solubility, fluorescence excitation/emission, etc. For the feasibility of your oligo conjugates, please contact [email protected].

Our standard method is desalting. When additional purification is requested or needed, we perform HPLC—reverse phase (RP) being the most common. Depending on the sequence and modifications, we may perform anion exchange (AX) and ion pairing (IR) HPLC. If you need other purification methods, please submit your inquiry at trilinkbiotech.com/oligobuilder or contact us at [email protected].

Dyes listed with the "extra purification step" requirement are made in a two-step process. First, the unlabeled oligonucleotide with the appropriate amino or thiol linker is synthesized and then purified to isolate only the full-length oligonucleotide. Then the oligonucleotide is conjugated to the dye and purified again to remove any unlabeled material.

Some fluorescent dyes, such as 6-FAM, TET and HEX, do not require this extra purification step. These dyes are available as phosphoramidite reagents, allowing us to label the oligonucleotide during the synthesis procedure and use a single purification step.

Oligonucleotides are synthesized according to the scale you requested. However, the final products have a lower yield than their starting scale. This is because byproducts are formed and some synthesizing materials are lost as each synthesis goes through several steps.

If you need a defined amount of an oligonucleotide, please specify it on your order at trilinkbiotech.com/oligobuilder.

The final yield of oligo synthesis can be impacted by many factors, including oligo length, modifications, and purification methods. Even when the same oligo is synthesized on the same scale, the final yield may vary due to contaminants, environmental factors (e.g., humidity), and synthesis efficiency. If you need a defined amount of an oligonucleotide, please specify it on your order at trilinkbiotech.com/oligobuilder.

We confirm quality of our oligonucleotides by mass spectrometry and PAGE. Add-on analytical services—e.g., HPLC, gel densitometry, dye spectrum, conductivity, enzymatic digest, bioburden assays, etc.—can be requested for additional cost during your quote inquiry.

For complex oligonucleotides that require mass spectral analysis not available at TriLink, you may request mass spec at the time of quote for an additional fee. For mid-scale (50 mg or greater) syntheses, we also perform HPLC analysis at no additional cost.

We supply a certificate of analysis with our oligonucleotides. The certificate contains the sequence info, molecular weight, calculated extinction coefficient, modifications, yield, purity, and QC data.

The optical density unit (OD260 unit) is a spectrophotometric measurement used to quantify oligonucleotides. This standardized unit represents the quantity of oligonucleotide required to produce an absorbance reading of 1.0 at a wavelength of 260 nm in 1 mL of solution using a 1 cm light path.

Since the sequence and composition of a particular oligonucleotide are known, the OD260 unit serves as a reliable way to quantify oligonucleotides. Absorbance values are commonly used to measure the mass of an oligonucleotide and aliquot it to a desired amount.

The extinction coefficients of the single strands are added together to provide the final data on our certificate of analysis for the duplex. When oligonucleotides are annealed, however, their individual ODs are not entirely additive; the duplex’s extinction coefficients can be 15-25% lower than the added value due to the hyperchromicity of the duplex.

This is generally not an indication of impurity. A thiol-modified oligonucleotide that is not completely reduced will be in three states:

1. Free thiol (oligonucleotide-SH)
2. Protected thiol (oligonucleotide-S-S-C6-O-DMT) and
3. Dimerized oligonucleotide (oligonucleotide-S-S-oligonucleotide).

We recommend that you reduce these oligonucleotides fully—preferably with DTT or TCEP as reductants—just prior to analysis to ensure that only the free thiol is present.

We lyophilize our oligonucleotides in a lyophilizer or speed vac.

Our oligos are typically shipped at room temperature or per your requested conditions.  Upon receipt, your oligo should be kept at -20° C for long-term storage.