Anneal Oligonucleotides
Last updated
Last updated
This protocol is for annealing two single-stranded oligonucleotides with complementary sequences (Figure 1). Heating followed by slow 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.
Pre-heat hot plate to max temperature.
Re-suspend each oligonucleotide to 100 uM in ultrapure water.
Combine 5 uL of each oligo, and add ddH2O to 100 uL final volume.
Bring 500 mL of water in a 1 L beaker to a boil in the microwave. Move to hot plate. Add magnetic stir bar and gently stir.
Place your oligo-containing tubes in a floating tube rack. Float in boiling water for 3 minutes.
Place beaker of water + tubes in styrofoam box. Let cool overnight (or at least 8 hours). Oligo concentration is 5 uM.
To ligate into plasmid vector: Dilute annealed oligos by 10X. Add 10 ul of annealed oligos to 90ul ultrapure water. The concentration is now 0.5 uM or 500 fmoles / ul.
Check out the Annealing oligonucleodies protocol by Integrated DNA Technologies.
