Building dsDNA with Oligos

  1. Prepare 10X annealing buffer (1M NaCl; 100mM Tris-HCl, pH 7.4). This will be done for you.
  2. Design oligos using the "Oligator". You do this in lab.
  3. Synthesize DNA oligos (a company will do this for you). Store -20 C. We prefer to have the company produce them at 100 M.
  4. Mix all the oligos and buffer (1X final concentration) for a total volume of 20 L. Use 0.5 mL microfuge tube.
  5. The final concentration of each oligo should be 5 M, so add 1 L of each oligo stock to the mixture with a final volume of 20 L.
  6. Boil in water (~500-600 mL volume) for 10 min. Turn off flame and allow to cool slowly in the hot water bath. This should take many hours, so overnight is a good time window.
  7. If sticky ends were designed, go directly to ligation. Otherwise, digest and proceed accordingly.
  8. You could directly to ligation and use the normal ligation formula to determine how many nanograms you want for your ligation.
  9. An easier approach is to approximate the dilution by diluting your annealed oligos 100 fold. Once the annealed oligos are diluted 100 fold, you can add 1 L of the assembled oligos into the ligation.

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