Rapid HPLC™ DNA Oligos

Purification removes truncated products and other synthesis impurities. Our experience has shown that purifying an oligonucleotide that will be used in demanding applications saves both time and money in the long run. We recommend considering additional purification for any oligonucleotide that will be used for an application other than routine PCR, qPCR, or Sanger DNA sequencing.

As a general rule, IDT also recommends that any oligonucleotide longer than 40 bases should receive further purification. For questions, please contact us.

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Customers who are new to working with large amounts of material are sometimes surprised when they see a yellow or brown discoloration to their oligos. If you have experienced this, do not worry. Color variation is normal in custom synthesis, especially as concentration increases. Importantly, IDT does not expect this to affect oligo function in our customers' experimental applications.

The image above depicts normal color variation as seen in oligo pools which are formulated to the same concentration (200 nmol oligos in 15 mL solution). In general, modified oligos at larger scales are more likely to display the variation seen above, while small-scale unmodified oligos (e.g., 25 nmol) tend to remain colorless in solution.

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The sodium salt exchange we perform post-HPLC purification is a sodium acetate precipitation. It helps remove any remaining TEAA in the oligo preparation after HPLC purification.

It is important to make sure oligos used in in vivo assays are TEAA-free because it can be toxic to cells.

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Oligo synthesis is accomplished through a series of steps, including coupling of individual bases, cleaving the oligo from the solid support, desalting, and if requested, purification of the oligo by HPLC or PAGE. No chemical reaction occurs with 100% efficiency, and, thus, each of these steps will incur a loss of final yield, which varies from specific sequence synthesis to synthesis.

Due to this variation, IDT custom oligos are ordered according to the amount of starting material used for the synthesis, referred to as the scale. While we cannot predict the actual final yield, we do guarantee a specific minimum yield for each oligo based on its sequence, starting scale, and the typical yield obtained under those specified conditions.

If you would like to know the minimum guaranteed yield for a specific oligo, simply add it to your Shopping Cart. The Shopping Cart provides you with the guaranteed minimum yield. If it is insufficient or excessive for your needs, simply edit the scale.

Please contact us if you have any questions about the yield you have received.

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Standard DNA oligos are synthesized up to 100 bases, while our high fidelity Ultramer™ Oligonucleotides can provide single-stranded DNA up to 200 bases.

Beyond 200 bases, we offer our new gBlocks™ Gene Fragments which are double-stranded DNA constructs available from 126 bp up to 2000 bp.

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Partial shipping means that we are shipping the oligos you ordered to you as they are completed, rather than our default method of waiting to ship until the entire order is complete, with all of the oligos shipping at the same time. We make every effort to synthesize and ship your oligos as quickly as possible because we know that they are time sensitive.

On occasion, one or more oligos may become delayed, e.g., because they did not pass one of our quality control checks or their processing took longer than anticipated. In these cases, we can ship you a partial order at your request. You can also choose to have your order ship partial if you know some of your oligos will take longer than others (because of purification, etc.)—such requests will incur an extra shipping charge.

If you would like to set an order to ship partial, you can request it at the time of checkout or  contact us.

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IDT offers a free online calculator, the OligoAnalyzer™ Tool, that will determine the T_m of your oligonucleotide sequence for reaction conditions you specify.

Be sure to include the Mg²⁺ and dNTP concentrations you plan to use in your reaction, as this will affect the T_m for your oligo.

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Standard desalted oligos can be used in routine PCR, qPCR, and DNA sequencing applications.

Through improvements to the traditional phosphoramidite chemistry and advances in instrumentation, IDT has obtained a coupling efficiency that allows standard desalt oligos to work well for these applications.

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We do not do post-synthesis sequencing of oligonucleotides. However, the oligos are checked by mass spectrometry to check that the oligos have the correct molecular weight.

The products we perform post-synthesis sequencing on are the double-stranded gBlocks™ Gene Fragments, and the Gene and Mini Gene™ synthetic genes.

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Although it may be possible to anneal oligos at room temperature, heating to denature the oligos and then cooling slowly to anneal the two oligos will help to ensure more efficient annealing and favor the stable duplex formation.

If you choose not to heat the oligos, it would be prudent to carefully screen the oligos for secondary structures which could interfere with the annealing reaction.

More information on how to anneal oligos can be found in the Annealing Oligos DECODED™ article.

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Though the majority of product will be the desired sequence, standard desalt oligos will contain a heterogeneous population of sequences, and this heterogeneity will increase with increasing length of the oligo. Oligos are synthesized 3'->5', base by base, through a series of chemical reactions. Chemical reactions are rarely, if ever, 100% efficient; the coupling reaction itself is approximately 99.6% efficient. With each base addition, less than 1% of the growing oligonucleotide chains will not undergo base extension.

After base coupling, we perform a capping step to prevent any truncated molecules from participating in further base addition. Because the capping reaction is slightly less than 100% efficient, a very small percentage of the truncated mutants will remain uncapped and can react during subsequent couplings steps. This leads to the formation of deletion mutants. The resulting oligonucleotide preparation, while comprised mostly of the ordered sequence, is a mixture of the full length oligos, truncated sequences, and sequences with internal deletions.

For exceptionally long oligos, such as our Ultramer™ Oligonucleotides and xGen™ Hyb Panels, IDT uses a proprietary IDT Ultramer synthesis process, which has been designed to give a high coupling efficiency (99.6%). This high coupling rate helps to allow us to synthesize long oligos (xGen Custom Hyb Panels are 60–120 bases long) with a high percentage of full-length product.

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For long term oligo storage, temperature is the most important factor to consider. For long term storage, whether oligos are dried down, or resuspended in non-DEPC treated water or TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE), we recommend that you store your oligos at –20°C.

For 4°C storage, oligos are stable for at least 60 weeks when stored dry, or resuspended in non-DEPC treated water or TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE).

It is also important to note that for DNA oligos, an acidic pH can induce depurination, therefore maintaining a neutral pH is important. For RNA oligos a neutral pH is also important, as basic pH buffers can induce RNA breakdown.

Please see the DECODED, Storing oligos: 7 things you should know, for data on oligonucleotide storage and a more thorough explanation.

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Oligonucleotides provided by IDT are desalted—free of added salt. First, no inorganic salts, such as sodium or magnesium, are used when DNA is synthesized chemically. Our standard desalting step refers to the removal of small organic molecules left over from synthesis.

The desalting process does remove the majority of these organic molecules, though trace amounts can still be present in a shipped oligo.

For the most demanding applications sensitive to even trace amounts of salts, e.g., NMR or crystallography, upon receipt we recommend dialyzing your oligos or using a size exclusion column as a clean-up step.

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All of the custom DNA and RNA oligonucleotides that IDT offers are synthesized chemically, not enzymatically. As a result, there should not be DNase or RNase present.

It is worth noting that there are abundant nucleases present in most environments, so good laboratory practice is very important to minimize potential exposure. IDT also offers RNaseAlert® and DNaseAlert™ substrates to help identify nuclease degradation.

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