Oligo Annealing Buffer (Duplex Buffer)

You can use one of the following buffers for annealing oligonucleotides.

Annealing buffer: 100 mM potassium acetate, 30 mM HEPES, pH 7.5.

This buffer will not interfere with your ligation reaction.

Oligo annealing protocol

1. Resuspend both complementary oligonucleotides at the same molar concentration using annealing buffer.
2. Mix equal volumes of both complementary oligos.
3. Dispense 100 µl aliquots of the mixed oligos into PCR tubes (500 µl size). Do not overlay the samples with oil. Place the tubes in a thermal cycler and run: (i) heat to 95 °C and hold for 2 minutes, then (ii) ramp cool to 25 °C over 45 minutes. Store at 4 °C, or at −20 °C for long-term storage.

Note: Alternative oligo annealing buffers are 1X ligase buffer, 1X kinase buffer, or 10 mM Tris (pH 7.5–8.0), 100 mM NaCl, 1 mM EDTA.

Good luck with your experiments!