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 95o C Heat Block for 3 minutes. 7. Remove the tube and let it cool to room temperature. (~10 minutes) 8. Vortex the mixture
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