Ultramer™ DNA Oligonucleotides

Calculating this conversion requires you to know the extinction coefficient for your sequence. With this, you can easily convert from your nanomole amount to ODs. To determine the extinction coefficient, you can analyze your sequence using IDT's free OligoAnalyzer™ Tool. Results from this analysis will also provide you with nmol/OD₂₆₀ and µg/OD₂₆₀ values.

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DNA with homology to the sequences flanking a double-stranded break (DSB) can serve as template for error-free homology directed-repair (HDR) of the DSB. The efficiency of HDR is determined by the concentration of donor DNA present at the time of repair, length of the homology arms, cell cycle, and activity of the endogenous repair systems in the particular cell [1]. These factors contribute to the high variability of HDR efficiency observed across different cell lines, and particularly in immortalized cells [2]. Typically, in replicating mammalian cells, donor arms are at least 500 bp in length [3]. However, it is important to determine the optimal HDR conditions for your cell line.

Inserts between the homology arms are frequently in the 1–2 kb range [4]. While longer inserts are possible, the efficiency of recombination decreases as the insert size increases [5]. Finding successfully integrated inserts is likely to be challenging when inserts are greater than 3 kb in most mammalian cells.

Single-stranded oligo DNA (ssODN) has recently been identified as a substrate that is preferred by the HDR mechanism and often achieves good efficiency with homology arms as short as 40 bp [6,7]. The drawback to using ssODNs is that they are limited in length to a few hundred bases, so the insert size is limited. When using Ultramer^® oligos as templates for a short insertion, tag, or SNP conversion, we have found arm lengths of 30–60 nt to be sufficient.

References

1. Elliott B, Richardson C, et al. (1998) Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol, 18(1):93–101.
2. Lin S, Staahl BT, et al. (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3:e04766.
3. Thomas KR, Folger KR, Capecchi MR (1986) High frequency targeting of genes to specific sites in the mammalian genome. Cell, 44(3):419–428.
4. Dickinson DJ, Ward JD, et al. (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods,10(10):1028–1034.
5. Li K, Wang G, et al. (2014) Optimization of genome engineering approaches with the CRISPR/Cas9 system. PloS One, 9(8):e105779.
6. Chen F, Pruett-Miller SM, et al. (2011) High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods, 8(9):753–755.
7. Davis L and Maizels N (2014) Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair. Proc Natl Acad Sci U S A, 111(10):E924–932.

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Yes. If you can provide the aptamer sequence(s), you can order directly from the IDT website.

To order online click on Custom DNA oligos, or if your sequence contains RNA bases, Custom RNA oligos.

Enter your desired scale, sequence, and purification.

If you are ordering aptamers with a fixed sequence, IDT recommends HPLC or PAGE purification. For aptamer libraries containing random bases, IDT recommends standard desalt.

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Oligonucleotide can be lost due to adsorption into the tube wall when storing the DNA in polystyrene. We recommend using polypropylene tubes for oligonucleotide storage to minimize such loss.

All IDT oligonucleotides are shipped out in low absorption polypropylene tubes which can be used for long term storage without significant loss of material.

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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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Every IDT custom oligo is synthesized to order through a series of tightly controlled steps, including the coupling of the individual bases, cleaving the oligo from the solid support, desalting, and, if requested, purification. Each of these steps will result in a loss of final yield, which varies with each synthesis.

Due to this variation, IDT oligos are ordered according to the amount of starting material, also referred to as the scale. All custom oligos are assigned a minimum yield guarantee, but lot-to-lot variability in the final delivered quantity should be expected even when reordering the same sequence. For further questions about this subject, contact us.

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Proofreading polymerases (Pfu, Diamond Taq®, etc) have been shown to remove 3' bases from PCR primers [1].

If you experience this problem, you can protect your primer from degradation by adding one or more phosphorothioate bonds to the 3' end of your primer.

Also make sure to add the enzyme last, after the addition of dNTPs, in your PCR to minimize the risk of this issue taking place.

Reference

1. Skerra A. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. 1992;20(14):3551-3554.

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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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IDT oligos have various estimated ship dates depending on product type and composition. Oligos added to the shopping cart will show an estimated ship date for each line item.

Once the order is placed, this estimated ship date can be found on the order confirmation email as well as in the order history of your web account. You can find the order history at idtdna.com/site/orderstatus/orderstatus.

Select the order number you wish to review, and the status and estimated ship date of each line item will be shown.

IDT will email you a confirmation when your order has been shipped. This shipping confirmation will include the tracking number of the package, which you can use to monitor the status of your shipment. If you have additional questions about your order, please contact us.

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To determine the relative T_m of primers with non-complementary overhangs, only the complementary region should be taken into account.

You can obtain the T_m using the free, online OligoAnalyzer™ Tool.

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The T_m value reported on our spec sheets uses a 25 µM oligonucleotide and a 50 mM salt concentration. It does not take into account dNTP or Mg²⁺  concentrations.

To get an accurate T_m for your specific application, all of these concentrations should be entered and adjusted to match the reaction conditions you plan to use.

The free IDT OligoAnalyzer™ calculates T_m for the concentrations you input.

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IDT uses a proprietary desalting technique that removes some truncation products and small organic contaminants from the synthesized oligonucleotide preparation. Removal of n-1mers produced during synthesis requires additional PAGE or HPLC purification.

PAGE is recommended for unmodified oligos >80−100 bases, while HPLC is the preferred method for oligos modified with either fluorescent dyes or attachment chemistries.

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RNA is inherently less stable than DNA due to its chemical structure. Additionally, RNases are more prevalent in standard laboratory conditions than DNases.

As even the slightest exposure to RNase can impact RNA stability, IDT has not performed rigorous long term stability studies for RNA.

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The shelf life of an oligo is dependent on the temperature at which the oligo is stored and how the oligo is resuspended. Temperature is the more important of the 2 variables. Generally, oligos should be stored at –20°C. At this temperature an oligo has a minimum shelf life of 2 years, whether it is stored dry/lyophilized, in TE buffer, or in (non-DEPC treated) water.

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

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IDT recommends you aim for primers between 18–30 bases; however, the most important considerations for primer design should be their T_m value and sequence specificity. Primers should also be free of strong secondary structures and self-complementarity.

The free IDT OligoAnalyzer™ Tool can be used to evaluate these features.

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Standard DNA oligos have a coupling efficiency of 99.4%. Ultramer oligos have a 99.6% coupling efficiency. This increase results in higher fidelity synthesis. Regardless of synthesis choice, purification may be recommended for longer oligos based on your specific application.

For further information regarding Ultramer oligos, please see our Ultramer DNA Oligos catalog page.

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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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For most applications, it is best to purify PCR products by gel electrophoresis.

This simple method not only purifies the final PCR product but can be a valuable troubleshooting tool as well since nonspecific PCR products, primer-dimer products, negative amplification, and other elements can easily be identified by gel analysis.

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CE, short for capillary electrophoresis, is a purity measurement utilizing a gel filled capillary to assess the proportion of full-length products to truncated products present in the final oligo sample. HPLC or PAGE purified oligos up to 60 bases in length receive a complimentary CE trace to document final purity.

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The heterocyclic ring structures in DNA and RNA absorb light with a maximum absorbance near 260 nanometers (nm). An OD₂₆₀, or optical density 260, is defined as the amount of light at a 260 nm wavelength which will be absorbed by an oligo resuspended in 1 mL water and the concentration is read in a 1 cm quartz cuvette.

This method of measurement is considered the most accurate means of assessing the amount of oligonucleotide present following synthesis. The relationship between measured OD₂₆₀, molar extinction coefficient (ε260), and oligonucleotide concentration is given as: OD₂₆₀ = ε260 x concentration.

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