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Annealing oligo dNTP linkers

  1. Mix the following:
    • 45 μl of each linker strand dissolved in H2O at 10 pmol/μl
    • 10 μl of 10x Annealing buffer
      • 250 mM Tris-HCl, pH 8.0
      • 100 mM MgCl2
  2. Boil a beaker of water. Turn off the flame, and put the tube in the water.
  3. Allow to cool to room temperature.

Use about 0.5 μl for a 10 μl ligation.

I have had problems sometimes with this mixture inhibiting ligation. I that case, ethanol precipitation (1/10 volume NaAc, 2.5 volume EtOH) followed by resuspension in TE solved the problem.