Oligo’s at a high concentration (e.g. 100-500µM) anneal more efficient compared to lower concentrations. To anneal oligonucleotides, a salt is needed for proper hybridization. Most annealing buffers make use of 50mM NaCL in TE. If a thermocycler is available, a program of heating at 94°C for 2 min and decreasing 1°C per minute till room temperature is a save method for demanding sequences (hairpins and high GC content). Otherwise heating up a heatblock or water bath till 94°C, insert the oligonucleotide mixture, plugging out the system and allow to cool till room temperature is also a verified method. These methods works for most nucleotide types (e.g. RNA, LNA, Chimera’s), backbone modifications (e.g. Phosphorotioates) and other modifications.  

Download our Product and Services Brochure 2022-2023 to learn more!

Products 

• SARS-CoV-2 Solutions - Primers & Probes
• DNA Synthesis (2-200+ nt)
• Probes for qPCR
• RNA Snthesis (Synthego partnership)

Services:

• Custom Plate Synthesis 
• Oligo Dispensing Services (pre-mixed assays, custom buffers or concentrations, specific plastic wear all possible!)
• Custom Solutions! Let our experts help you.

Dual labelled oligonucleotides with a Quencher and a Fluorophore for e.g. qPCR are a separate product class at Biolegio. These oligonucleotides are per default HPLC purified and assessed for quality on UPLC-MS to meet our high quality standards. They can be ordered as:

• TDP: Twice Dyed Probes. These hydrolysis probes are the equivalent of Taqman probes used in qPCR. For more information on TDP’s click here.
• XS probes: These hydrolysis probes are the equivalent of MGB probes where the Tm of the probe is raised to increase specificity. For more information on XS probes click here.
• MB probe: Molecular Beacon Probes. For more information about these stem-loop structured probes click here.
• FRET probe: Fluorescent Resonance Energy Transfer probes for a.o. real-time PCR. For more information click here.       

There are several places where the properties of an oligo can be found.

• In the webshop, length, melting temperature (TM) and GC% are displayed while the sequence is entered.
• Once the oligo is shipped, these numbers plus base composition, molecular extinction coefficient and molar mass can be found on the specifications.
• Specifications are shipped digital per default, printed if requested and are downloadable from the webshop at all times. 

To order mixed or degenerate nucleotides in a sequence, you can enter these according to the IUPAC code (https://www.bioinformatics.org/sms/iupac.html). As a standard these positions will be machine-mixed; that is the synthesizer mixes the correct ratio’s during synthesis. On average a hand-mixed solution can give a slightly more accurate distribution. For custom ratio’s or hand-mixed positions please contact Biolegio at info@biolegio.com.

In the synthesis-labelling-purification and QC methods we don’t rely on enzymatic processes so there are no enzymes or proteins present. We synthesize and process our oligo’s in a clean and controlled environment where contamination by enzymes/proteins present in most human environments is minimized. 

TE buffer has proven to be the most stable method for storing oligo’s. If other storage/shipment conditions are preferred you can choose other standard conditions or contact Biolegio at info@biolegio.com.

Biolegio offers three purification options: Standard, HPLC and PAGE.

• Standard: the standard purification is a desalt purification. Please read "Do standard oligo's receive a purification step such as desalting"?
• HPLC: Oligo's that require a higher purity for their application can be purified by HPLC. This will remove the majority of truncated sequences and salts inherent to chemical synthesis of oligo's. The addition of modifications often require HPLC purification since the coupling efficiency of modifications on average is much lower compared to the standard nucleotides. Some modifications require 2x HPLC which is performed per default for these modifications. For some products (such as dual labelled qPCR probes) the HPLC step is default and included in the price calculation. 
• PAGE: to reduce the maximum amount of impurities PAGE purification is applied. Tis is a time-consuming and thus more expensive option. The more since the yield will be significantly lowered. HPLC looses resolution for longer (roughly > 50 nts) oligo's and longer oligo's for e.g. cloning are recommanded to be purified by PAGE.  

In doubt if your oligo requires purification? Please contact info@biolegio.com and our product specialists can advice you on this subject.   

A major challenge in many oligo based applications is their susceptibility to attack by a variety of nucleases present in cells and body fluids such as blood, urine and saliva. There are several modifications available to protect your oligo's:

• Phosphorotioates (PTO): the double bonded non-bridging oxygen atom in the phosphate backbone is replaced by a sulfur atom. The PTO prevents the oligo from enzymatic degradation and can be used throughout the sequence. The synthesis results in two isomeric forms and only one conformation is effective against degradation. The synthesis can’t be purposely directed to yield one of the two isomeric configurations. To state it very abstract: it is a 50/50 chance to achieve a functional conformation. So introducing one PTO on the 3’side will protect 50% of the oligo for 3'exonuclease activity (e.g. ... GTCGT*T-3’). Introducing a second PTO one base upstream (e.g. ... GTCG*T*T-3’) will protect 75% of the oligo, a third (e.g. ... GTC*G*T*T-3’) will protect 87.5% of the oligo etc. 
• 3' and 5' modifications: almost all modifications available will protect the oligo from exonuclease activity by some extend. 
• The storage medium: a fact often overlooked is the medium in which the oligo is stored. The stability of an oligo, and the more of certain modifications, is pH and temperature dependend. The best practise for oligo stability is to store the oligo on a low temperature (at least refridgerated) in a buffered solution.    

To increase multiplexing capacity in NGS analysis, it is crucial to mitigate cross-contamination of oligo's containing index sequences. Biolegio has optimized the (post-)synthesis process to bring down the level of cross contamiantion below 0.1% using SASI-Seq. This means that, when ordering the index oligo's NGS-Standard or NGS-Premium grade, misassignement levels of an index to other reads are below 0.1%. 

For further reading about index cross-contamiation please visit our Knowledge base.  

Yes, Biolegio synthesized oligo's up to 300 nucleotides long with positive feedback from multiple customers. However, the succesfull use of long oligo's depend on the the application it is used for. 

The longer the oligo gets, the more difficult it is to maintain a high coupling efficiency and thus the more advisable to order the oligo purified depending on the application. Inherent synthesis failures accumulate during synthesis (see "coupling efficiency"). The coupling efficiency depends on multiple variables and sequence is a predominant variable.

Biolegio produces oligo's up to 200 nucleotides for many different applications. The length cut-off is based on customer feedback analysis and allthough we can synthesize longer oligo's we prefer to discuss your oligo needs before we start the synthesis. Please contact info@biolegio.com to contact our product specialists! 

Biolegio listed the most-ordered modifications in the webshop. If you can't find the modification you are looking for, please send an email to info@biolegio.com. Our produt specialists will review your request.

There are quite some guidelines to be found online. Biolegio has combined +20 years’ experience and  compiled the available information online leading to the following basic considerations:

• Oligo’s are best stored in a TE buffer and if a chelating/buffering agents are not desirable in the application a LowTE buffer or sterile water will be the best option. In general the following order of storage conditions is advised: TE/LowTE<lyophilized/dried<water.
• Storing oligo’s at -20° is optimal. Refrigerated oligo’s (<4°C) are stable for at least a year. Oligo’s stored at room temperature dried or dissolved in TE/LowTE buffer are stable for at least a month.
• UV light has detrimental effects on oligo’s so store them preferably in a dark place. This is especially true for oligo’s modified with fluorophores as they might bleach upon exposure to UV light. Besides this modified oligo’s can in general be treated as non-modified oligo’s for optimal storage conditions.