Annealing Two Single Stranded Oligos To Make Double Stranded DNA (i.e. How to make linkers)

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PROCEDURE
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1) Dissolve oligos in STE buffer
   • Buffer composition:
     - 10 mM Tris, pH 8.0
     - 50 mM NaCl
     - 1 mM EDTA
   • Note: The presence of salt is required for efficient hybridization.
   • Dissolve each oligo at high concentration:
     ~10 OD260 units per 100 µL.

2) Mix complementary strands
   • Combine the two strands in EQUAL MOLAR amounts.
   • Unequal molar ratios will result in residual single-stranded material.

3) Heat denaturation and annealing
   • Heat mixture to 94 °C.
   • Allow to cool gradually to room temperature.

   Cooling options:
   • Simple sequences:
     - Remove from heat and cool on the benchtop at room temperature.
   • Sequences with significant hairpin potential:
     - Use a gradual cooling step (e.g., water bath or temperature block).
     - Heat to 94 °C, then turn off/unplug the device and allow slow cooling.

4) Storage
   • The annealed product will be stable in double-stranded form.
   • Store at:
     - 4 °C for short-term use
     - Frozen for long-term storage

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THINGS TO CONSIDER
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• Ligation applications:
  - A 5′ phosphate may be required.
  - This can be added:
    - During oligo synthesis (chemical phosphorylation), or
    - Post-synthesis using PNK (enzymatic phosphorylation).

• Oligo length / cloning applications:
  - For longer oligos or cloning workflows, starting with PAGE-purified oligos
    is recommended for best results.