Determining the Physical Characteristics of Your Oligos
IDT offers a powerful analytical tool for determining the properties of oligo sequences—the OligoAnalyzer program. 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 program from under the “SciTools” heading of the IDT website (Figure 1, Arrow).
Figure 1. Select OligoAnalyzer From the IDT Website Main Page.
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® Program.
The OligoAnalyzer program 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).
A. Results of Dimer Analysis.
B. Results of Hairpin Analysis.
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.
Author: Stephanie Youtsey is a Technical Support Representative at IDT.