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Determining the physical characteristics of your oligos—the OligoAnalyzer Tool

IDT offers a powerful analytical tool for determining the properties of oligo sequences—the OligoAnalyzer® Tool. This tool calculates the physical characteristics of any oligo sequence, including length, GC content, melting temperature range, molecular weight, extinction coefficient, and optical density.

Access the OligoAnalyzer Tool from under the “Tools” heading on any page of the IDT website (Figure 1).

Figure 1. Select OligoAnalyzer® Tool from any page of the IDT website.

Enter your sequence into the “Sequence” box in the 5’ to 3’ orientation (Figure 2, Arrow 1).

Figure 2. Insert oligo on sequence entry page of OligoAnalyzer® Tool.

The OligoAnalyzer Tool accommodates DNA, RNA, and mixed bases, as well as a range of modifications, which can be selected from the menus under the Sequence box (Figure 2, Arrow 2).

To get the correct Tm, it is important to enter the Mg2+ and dNTP concentrations you will use in your experiment (Figure 2, Arrow 3). Please note that the Tm reported on the specification sheet shipped with IDT oligos uses default settings of 0 nM Mg2+ and 0 mM dNTPs, and thus, will usually differ from the Tm calculated here.

The “Analyze” button (Figure 2, Arrow 4) will produce the complementary sequence, GC content, melting temperature, molecular weight, and extinction coefficient.

You can also run Self-Dimer and Hairpin analyses of your sequence from the Sequence entry screen (Figure 2, selections below Arrow 4). The ΔG value (Figure 3, Panel A) gives an indication of the strength of the secondary structure. IDT recommends the ΔG value be more (less negative) than –9 for self-dimers and hetero-dimers. For hairpins, the Tm should be lower than the temperature at which the oligo will be used (Figure 3, Panel B).

Figure 3. Run self-dimer and hairpin analyses.

If you need help with the program, the “Instructions” link provides step-by-step guidance on how to use the OligoAnalyzer Tool. The “Definitions” link gives an explanation of terms and equations the program uses to determine Tm and extinction coefficient (Figure 4, Arrow). And, of course, you can always contact an IDT Customer Care Representative.

Figure 4. Get help with program and definitions.

Product focus—free web tools, and oligos, mods, and dsDNA

SciTools® Web Tools

Explore IDT SciTools Web Tools for free, online tools for oligonucleotide analysis and for qPCR probe and assay design. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm.

Custom Oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Learn more or order now.

Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to noncat@idtdna.com.

Custom dsDNA Fragments

Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.

Related reading

Calculation tips for resuspending and diluting nucleic acids—Use these simple guidelines for making a 100 µM solution; calculating nanomoles, micrograms, copy number, and concentration; and determining concentration equivalencies.

The importance of Tm in molecular biology applications—Learn how to predict and select appropriate Tms for oligo hybridization steps, including PCR.

Oligo quantification—getting it right—Did you know that a supplier's yield readings can differ from what the researcher calculates after resuspension? Learn the importance of using the [right] molar extinction coefficient in calculations of oligonucleotide concentration.

Review other DECODED Online newsletter articles on oligo handling and analysis.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Stephanie Youtsey is a Technical Support Representative at IDT.

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