Recommended solvents: The oligos can be resuspended in sterile nuclease-free water or preferably in TE buffer (10 mM Tri-HCl, 0.1 mM EDTA, pH 8.0) if storage of the oligo solution for extended periods of time is required. Concentration: To prepare a Stock solution at a concentration of 100µM: multiply by 10 the total number of nanomoles delivered (you will find it on the label and certificate of analysis of your oligo/probe). The resulting number will be the volume in microliters of solvent to use for resuspending. Example: Final yield: 23.2 nanomoles Volume of solvent needed to prepare Stock solution: (23.2)X(10)= 232 µl Resuspension: Briefly centrifuge your vials to ensure that the oligo pellet goes to the bottom. Use the recommended solvent or solution. Allow the oligo to hydrate for several minutes at room temperature, then vortex. Storage: Lyophilized oligos can be stored at -20°C or room temperature and are stable for long periods of time before being handled for the first time. It is important to resuspend the oligos in TE buffer or nuclease-free water to ensure physical integrity and avoid degradation. Resuspended oligos should be aliquoted and stored at 4°C or -20°C. Avoid freezing and thawing processes of the oligos solution to prevent degradation. When dealing with probes, protecting them from light is indispensable to avoid photobleaching.

There is often confusion surrounding the meanings of “synthesis scale” and “synthesis yield.” Simply put, scale refers to the amount of starting material used to synthesize the oligonucleotide. Yield, on the other hand, refers to the amount of oligo recovered after all stages of synthesis and purification. No chemical reaction or physical process is 100% efficient, so the final deliverable amount of oligonucleotide is never equal to the starting amount for synthesis. Factors such as oligo size, modifications, coupling efficiency, nucleotide composition, and additional purification processes further affect the amount of final product (yield).

T4 Oligo recommends using standard desalted oligos in routine PCR amplifications, qPCR and DNA sequencing processes. However, if your oligos are longer than 40 bases in length, we recommend additional purification in order to significantly improve the efficiency of your oligos.

T4 Oligo recommends purifying any oligo that is used for sensitive applications such as real-time PCR or DNA sequencing, as the purification process removes incomplete oligos that are normally generated during the synthesis process. A purification process significantly improves the efficiency of the oligos, especially with respect to the specificity of recognition of their target sequence.

No, due to yield loss during purification, the smallest scale allowed is 50 nanomoles in the case of cartridge and 100 nanomoles for PAGE or HPLC purification.

T4 Oligo recommends cartridge purification for short and long unmodified oligos, as well as for short degenerate oligos (10 to 65 bases) used for cloning, lagging and mutagenesis, among others. T4 Oligo recommends HPLC purification in the case of fluorescently labeled oligos since, due to their hydrophobicity, they are best purified by this method. An HPLC purification helps to remove by-products of the synthesis resulting in oligos and probes with higher specificity. We offer reverse phase HPLC purifications (RP-HPLC) in the case of fluorescently labeled oligos and/or other hydrophobic modifications. If you have any doubts or questions, do not hesitate to contact us at soporte@t4oligo.com, where the person in charge of the synthesis of your oligo will be available to assist you.

A purification by HPLC guarantees 85% purity, while a purification by electrophoresis (PAGE) guarantees 90% purity. In the case of purification by cartridge, a purification of about 90% is guaranteed.

A degenerate oligo is actually a collection of primers that vary in one or more bases called degenerates. For example, an oligo with an “N” degeneracy means that any of the 4 nucleotides (ATGC) can be present at that position, ideally in a ratio of 25% each. The usefulness of a degeneracy is directly dependent on retaining the ratio of variants, which can be altered by purification. The decision to purify an oligo is directly influenced by your research needs. A purification process will eliminate by-products originating from the synthesis process but will also modify the equimolar ratio of the oligos containing the different nucleotide variants. Taking this into account, there are some cases in which the purification of an oligo is justified to a greater extent, for example, the case of very long oligos or oligos modified near the 3′ end. Outside of these cases it is important to consider not purifying, as it may just be an unnecessary drain on your budget. If you have any questions about when to purify a degenerate oligo, please do not hesitate to contact us at ventas@t4oligo.com, where we will assign you a personalized specialist.

T4 Oligo recommends cartridge purification for short and long unmodified oligos, as well as for short degenerate oligos (10 to 65 bases) used for cloning, lagging and mutagenesis, among others. T4 Oligo recommends HPLC purification in the case of fluorescently labeled oligos since, due to their hydrophobicity, they are best purified by this method. An HPLC purification helps to remove by-products of the synthesis resulting in oligos and probes with higher specificity. We offer reverse phase HPLC purifications (RP-HPLC) in the case of fluorescently labeled oligos and/or other hydrophobic modifications. If you have any doubts or questions, do not hesitate to contact us at soporte@t4oligo.com, where the person in charge of the synthesis of your oligo will be available to assist you.

Please note that probes with sequences larger than 30 bases will probably not have as much quenching efficiency, for such cases we suggest to consider placing the quencher internally. Likewise, if you use previously reported sequences and they are smaller than 20 bases, they may be designed as Minor Groove Binder probes and probably will not be able to bind at the appropriate temperature of 70 °C. Contrary to what marketing campaigns indicate, it is not necessary to invest heavily in using probes containing the MGB™ molecule, T4 Oligo has private and accessible alternatives with similar or superior results.

A variety of PCR additives and some potentiating agents have been used to increase the yield, specificity and consistency of PCR reactions. While these additives may have beneficial effects on some amplifications it is impossible to predict which agents will be useful in a particular context and therefore must be tested empirically for each combination of template and primers. some of the most popular of these additives are described below. DMSO at 2-10%, may be necessary for amplification of some templates, however, concentrations greater than 10% DMSO can reduce Taq polymerase activity by up to 50%, so it is not recommended for routine use. DMSO is believed to reduce secondary structure and is particularly useful for GC-rich templates. Betaine or betaine monohydrate is generally used at a final concentration of 1.0 to 1.7 M. Formamide is generally used at 1-5%, and 10% formamide is reported to have no effect on Taq polymerase activity. Non-ionic detergents stabilize Taq polymerase and can also suppress secondary structure formation. 0.1-1% Triton X-100, Tween 20 or NP-40 can increase yield, but can also increase non-specific amplification. Concentration by SDS in the nucleic acid extraction procedure down to 0.01% can inhibit PCR by reducing Taq polymerase activity to a minimum of 10%, however, inclusion of 0.5% Tween 20 or NP-40 will effectively neutralize this effect. TMAC is generally used at a final concentration of 15-100 mM to remove the nonspecific primer. TMAC has also been used to reduce the potential DNA-RNA disparity and improve the stringency of hybridization reactions. The base analog 7-deaza-2’deoxyguanosine can facilitate amplification of templates with stable secondary structures when used in place of dGTP at a ratio of 3:1,7-deaza-2’deoxyguanosine. BSA has proven particularly useful when attempting to amplify old DNA or templates containing PCR inhibitors such as melanin. Formamide is generally used at 1-5%, and 10% formamide is reported to have no effect on Taq polymerase activity.