This protocol is for annealing two single-stranded oligonucleotides with complementary sequences (Figure 1). Heating followed by cooling facilitates hybridization.
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.
EDTA: Ethylenediaminetetraacetic acid
NaCl: Sodium Chloride
Trizma® base: Brand name for Tris [Tris(hydroxymethyl)aminomethane]
Oligo: Abbreviation of oligonucleotide or oligomer. Oligonucleotides are short, single-stranded DNA or RNA molecules that must be annealed (heated or melted) so they can bond and form a double strand with an appropriate complementary DNA or RNA strand.
DNA annealing: This page discusses the annealing process for all oligonucleotides. Sometimes annealing is referred to as DNA annealing even though the process is used for RNA as well. Annealing is the process of heating and cooling two single-stranded oligonucleotides with complementary sequences. Heat breaks all hydrogen bonds, and cooling allows new bonds to form between the sequences.
The annealing process is divided into two main steps: 1) dissolution, and 2) annealing, either by heat block or thermocycler.
Although each oligonucleotide comes in a measured amount, for best results, verify it with a spectrophotometer to ensure that equal amounts of each oligonucleotide are added to the reaction.
The desired concentration of the duplex oligonucleotide is 50 µM.
*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.
Although a heat block will work, a thermocycler allows for a more consistent process.
Following deployment of the heat block or thermocycler, the duplex oligonucleotide is now ready to use or stored. To learn more about storing oligonucleotides, see Handling Guidelines & Stability.
Make all buffers with Milli-Q® water.
This buffer is typically used with T4 DNA Ligase.
This buffer is typically used with T4 Polynucleotide Kinase.