Protocol for Annealing Oligonucleotides

1. Prepare a solution of 10X annealing buffer.

10 mM MgCl₂, 200 mM Tris-HCl, pH 8.0

2. Prepare an aliquot of the oligonucleotide probe in an eppendorf tube.

3. Add a molar excess of oligonucleotide target to the tube.

4. Calculate the volume of 10X annealing buffer to add.

Reaction Volume = Volume of Probe + Volume of Target

Volume of 10X Annealing Buffer = 9 x Reaction Volume

5. Add the 10 X Annealing buffer and vortex to mix.

6. Place the tube in a 95^oC Heat Block for 3 minutes.

7. Remove the tube and let it cool to room temperature. (~10 minutes)

8. Vortex the mixture