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Annealing Oligonucleotides

It is sometimes necessary to make double-stranded DNA from single-stranded, complementary oligonucleotides. While the annealing procedure is fairly straightforward, attention to a few details can greatly reduce the presence of undesired single-stranded material.


  1. Resuspend. After briefly spinning down each oligonucleotide pellet, dissolve in Duplex Buffer (100 mM Potassium Acetate; 30 mM HEPES, pH 7.5; available from IDT)—this provides a buffering environment and the salt is necessary for oligonucleotide hybridization. Dissolve each oligo at high concentration (1–10 OD260/100 μL); see Table 1 for guidelines on resuspension volumes. Heating (up to 94°C) and vortexing will facilitate resuspension.

  2. Mix. Add the 2 strands together in equal molar amounts. This step is critical to avoid residual single-stranded material.
  3. Anneal. Heat the mixed oligonucleotides to 94°C for 2 minutes and gradually cool. For many oligos "cooling" can be as simple as transferring samples from the heat block or water bath to the bench-top at room temperature. For sequences with significant secondary structure, a more gradual cooling/annealing step is beneficial. This is easily done by placing the oligo solution in a water bath or heat block and unplugging/turning off the machine.
  4. (Optional) Dilute. if needed, dilute the annealed oligonucleotides using Nuclease-Free Duplex Buffer or 1X IDTE Buffer (Part #11-01-02-05).
  5. Store. The resulting product will be in a stable, double-stranded form and can be stored at 4°C or frozen.

Things to consider:

Avoiding contamination and degradation—If you plan to use the duplex on multiple occasions, divide it into smaller aliquots and store at –20°C. Nuclease-free Duplex Buffer is available from IDT and is certified nuclease-free by testing with IDT RNaseAlert™ and DNaseAlert™ Kits. Find out more about these products at www.idtdna.com.

Secondary Structure—Low yields of the expected, annealed product can be caused by secondary structure. Use the OligoAnalyzer® program (www.idtdna.com/scitools) to determine whether there is significant secondary structure in the oligonucleotides. Problematic annealing can often be resolved by slow cooling, as described in Step 3, above.

Ligation—If the double-stranded oligonucleotide product will be used in a ligation reaction, you may need to add 5’-phosphates to the strand ends. These can be added at the time of oligo synthesis (chemical phosphorylation; done by request) or anytime thereafter (before or after annealing) using polynucleotide kinase (enzymatic phosphorylation).

Cloning—If the resulting double-stranded DNA fragment will be relatively long (>60 bp), or will be used in cloning, we recommend starting with PAGE-purified oligos (IDT can provide this service).

Annealing RNA—The IDT Research team also uses this protocol to create siRNA duplexes from single-stranded, complementary RNA oligos.

For additional information, please contact the Technical Support Group at applicationsupport@idtdna.com.

Ellen Prediger, PhD, is Director of Scientific Communications at IDT.

Now read: DNA Oligonulceotide Resuspension and Storage, or browse the content from the latest DECODED issue.