Annealing Oligonucleotides

    1. Determine the concentration of your oligonucleotide solutions. Dissolve lyophilized oligonucleotide in water or TE and verify concentration by determining the A260 value of duplicate dilutions. Use the conversion factor printed on the oligonucleotide label to determine nanomole/A260 unit

 

    1. Add the following components together:
      Stock Final Concentration
      Oligonucleotide 1 100 nanomoles/milliliter
      Oligonucleotide 2 100 nanomoles/milliliter
      * 10X Annealing Buffer 1X
      Nuclease-free water to appropriate volume

      * 10X Annealing Buffer: 100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA.

    2. Heat the oligo solution to a temperature 10° C higher than the calculated melting temperature. Maintain the temperature for 10 minutes. Remove the solution from the heating block/water bath and allow it to cool slowly to room temperature on the bench (approximately 1 hour).

 

  1. Store the annealed oligos at -20° C.
Copyright 2017 © Midland Certified Reagent Company