Annealing complementary Oligos

Protocol overview

1. Mix concentrated complementary oligonucleotides together at a 1:1 molar ratio in a microcentrifuge tube.
2. Dilute oligonucleotide mixture to a final concentration of 1 pmol/µl with a Tris or phosphate buffer containing salt; for  example, 10 mM Tris, 1 mM EDTA, 50 mM NaCl (pH 8.0) or 100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA (pH 7.5).  
3. Anneal oligonucleotides using one of the annealing methods described below.  
4. Aliquot and store probe at -20C. Alternatively, the double-stranded DNA probe may be stored at 4 for several weeks if protected from nucleases.  

Alternative Annealing Methods  

Option 1: Anneal with a heating block  
1. Incubate the oligonucleotides at 95 C for 5 minutes.  
2. Gradually reduce the heat until the oligonucleotides have reached room temperature.

Option 2: Anneal with a water bath  
1. Boil 400 ml of water in a large glass beaker on a hotplate.  
2. Incubate the tube of oligonucleotides in the boiling water for 5 minutes.  
3. Turn off the hotplate, leaving the oligonucleotides in the beaker on the hotplate to slowly cool to room temperature.    

Option 3: Anneal with a thermocycler
A temperature thermocyler enables convenient and reproducible annealing of oligonucleotides. Use the following table as a guide to program a thermocycler for either a simple or advanced protocol. The notation “-1C/cycle” indicates that the temperature of the heating block will decrease 1C per cycle.