All your questions in one place
Since Biolegio started in 1996, we received many questions and feedback regarding a wide range of subjects. This information has been consolidated into our Knowledge base and in our Frequently Asked Questions.
The content is still under construction at the moment and updated regularly. To provide you efficiently with the information you could be looking for, the FAQ's are structured in catagories. If you can't find the information you are looking for, please feel free to contact us by email at firstname.lastname@example.org.
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.
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.
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 email@example.com. Our produt specialists will review your request.
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 firstname.lastname@example.org to contact our product specialists!
At Biolegio we considere different kinds of contamination. This section regards the contamination of ssDNA sequences for amplification techniques like PCR. For information regarding cross-contamination of NGS indices (index hopping) please read "How does Biolegio control NGS index cross-contamination".
Oligo's are synthesized in relaitive high quantities. Where in general labs tubes containing 10E+6 (ss)DNA copies are considered a contamination risk, standard oligo providers work with quantities in the range of 10E+14 per oligo! Cost-effective oligo synthesis is performed on DNA synthesizers each producing up to several hundreds oligo's per day. The production of longer oligo's that can serve as a template for amplification is a realistic risk in this proces.
The best way to prevent the synthesis of high-risk oligo sequences is to not produce them. Every oligo sequence that enters our facility in silico is interrogated on contamination risk before further processing is made possible. If a sequence is considered a high risk, we contact the customer to discuss the options. This method has proven itself significantly during the SARS-CoV-2 pandamic where the request of contamination-free primers and probes for SARS-CoV-2 detection and analysis is of highest importance (for further reading regarding this subject please visit https://wwwnc.cdc.gov/eid/article/26/8/20-1843_article).
To maintain a clean lab Biolegio performs qPCR analysis on swabs taken throughout the facility. A stringent cleaning protocol is in place when new sequences that have been produced are added to the sequence interrogator. This database of high-risk sequencs is well established and growing based on customer feedback. Please contact email@example.com for further requests regarding high-risk sequences and secure your analysis!
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.
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.
All standard oligo's are desalted as a standard. During the post-synthesis process where oligo's are treated to become biologically active, salt are formed that can act as, for example, a PCR inhibitor. Biolegio uses a propriety desalt method that has been selected and validated by PCR efficiency analysis.
Note that certain applications such as cloning may or will need additional purification. To read more about this please read "Which purification methods does Biolegio offer?".
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 firstname.lastname@example.org and our product specialists can advice you on this subject.
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 email@example.com.
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.
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 firstname.lastname@example.org.
In our webshop we offer two different standard concentrations; 100µM (or 100pmol/µl) and 1nM (or 1nmol/µl). These are the most requested and ordered concentrations in >20 years oligonucleotide synthesis at Biolegio. The default shipping conditions as handled at Biolegio:
- Oligo's are shipped on a 100µM concentration.
- If the yield of an oligo to be delivered in a 2 ml tube exceeds 180 nmol, the oligo is shipped on a 1nM concentration.
However, custom concentrations are often requested. Please visit our website to read about our Dispensing Services or contact Biolegio at email@example.com.
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.
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.
The storage conditions determine the stability of oligo's (see "How should I store my oligo's"). Biolegio guarentees the quality of an oligo for at least one year. Allthough properly stored non-modified oligo's will be stable for many years, modifications such as fluorophores have a lower stability.
Download our Product and Services Brochure 2022-2023 to learn more!
- SARS-CoV-2 Solutions - Primers & Probes
- DNA Synthesis (2-200+ nt)
- Probes for qPCR
- RNA Snthesis (Synthego partnership)
- 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.