Protocol for Annealing Oligonucleotides

This protocol is for annealing two single-stranded oligonucleotides with complementary sequences (Figure 1). Heating followed by cooling facilitates hybridization.

Example of an annealing reaction

Figure 1. Example of an annealing reaction. Heat ‘breaks’ all hydrogen bonds, thereby disrupting any secondary structure within each oligonucleotide. Slow cooling then facilitates hybridization as new hydrogen bonds form between the complementary sequences.

Definitions / Abbreviations

  • EDTA - Ethylenediaminetetraacetic acid
  • NaCl - Sodium Chloride
  • Trizma® - Brand name for Tris [Tris(hydroxymethyl)aminomethane]

Equipment

  • Heat block or
  • Thermocycler

Supplies

  • 2 mL centrifuge tubes
  • Pipette tips
  • Milli-Q® H2O
  • EDTA (Sigma-Aldrich catalog #E9884)
  • NaCl (Sigma-Aldrich catalog #S3014)
  • Trizma® (Sigma-Aldrich Catalog #93362)
  • Two single-stranded oligonucleotides with complementary sequences

Method

The annealing process is divided into two main steps: 1) dissolution, and 2) annealing, either by heat block or thermocycler.

Dissolution

Although each oligonucleotide comes in a measured amount, we recommend verifying it with a spectrophotometer. This will ensure that equal amounts of each oligonucleotide are added to the reaction.

  • Dissolve each oligonucleotide in a volume of Annealing Buffer (see Additional Notes) so that each has the same concentration.
  • The concentration of each oligonucleotide needs to be 2X the desired concentration of the duplex oligonucleotide.

Example

The desired concentration of the duplex oligonucleotide is 50 µM.

  1. Oligonucleotide 1: delivered with 10.55 OD (312.6 µg, 49.9 nmol); verify measured OD amount with spectrophotometer.
  2. Oligonucleotide 2: delivered with 9.04 OD (279.7 µg, 45.9 nmol); verify measured OD amount with spectrophotometer.
  3. Each oligonucleotide stock solution needs to be 2X the desired duplex oligonucleotide concentration, i.e. each stock solution needs to be 100 µM.
    a.    *For oligonucleotide 1, add 49.9 x 10 = 499 µL of Annealing Buffer to create a 100 µM stock solution.
    b.    For oligonucleotide 2, add 45.9 x 10 = 459 µL of Annealing Buffer to create a 100 µM stock solution.

*This calculation is a shortcut that only works for creating 100 µM solutions and is used here for example purposes only. To learn more about calculating different oligonucleotide concentrations, see Handling Guidelines & Stability.

Annealing

Heat Block

  1. Mix equal volumes of the equimolar oligonucleotides in a microtube.
  2. Incubate the microtube at 95 °C for 5 min.
  3. Allow the microtube to slowly cool to room temperature (should take <60 min).

Thermocycler

Although a heat block will work, a thermocycler allows for a more consistent process.

  1. Mix equal volumes of the equimolar oligonucleotides in a PCR tube.
  2. Use the following thermal profile:
    a.    Heat to 95 °C and maintain the temperature for 2 min.
    b.    Cool to 25 °C over 45 min.
    c.    Cool to 4 °C for temporary storage.
  3. Centrifuge the PCR tube briefly to draw all moisture away from the lid.

Following deployment of the heat block or thermocycler, the duplex oligonucleotide is now ready to use, or alternatively, it can be stored. To learn more about storing oligonucleotides, see Handling Guidelines & Stability.

Additional Notes

The Annealing Buffer is what we use in our internal processes. The Ligase and Kinase Buffers are examples of what is commercially available. Make all buffers with Milli-Q water.

Annealing Buffer Composition (1X)

  • 10 mM Tris, pH 7.5 - 8.0
  • 50 mM NaCl
  • 1 mM EDTA

Ligase Buffer Composition (1X)

This is typically used with T4 DNA Ligase.

  • 50 mM Tris-HCl, pH 7.5
  • 10 mM MgCl2
  • 1 mM ATP
  • 10 mM DTT

Kinase Buffer Composition (1X)

This is typically used with T4 Polynucleotide Kinase.

  • 70 mM Tris-HCl, pH 7.6
  • 10 mM MgCl2
  • 5 mM DTT

 

Materials