Building dsDNA with Oligos

1. Prepare 10X annealing buffer (1M NaCl; 100mM Tris-HCl, pH 7.4)
2. Design oligos using the "Oligator".
3. Synthesize DNA oligos. Make sure the oligos are equal Molar concentrations. Store -20° C. We prefer to have the company produce them at 100 µM.
4. Mix all the oligos and buffer (1X final concentration) for a final volume of 20 µL. Use 0.5 mL microfuge tube.
5. The final concentration of each oligo should be 5 µM, so add 1 µL of each oligo stock to the mixture with a final volume of 20 µL.
6. Boil in water (~500-600 mL volume) for 10 min. Turn off flame and allow to cool slowly on bench top. 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 use the Loligator web tool. You will need to use information that came with your oligos (specifically total number of µg and volume in µL). The boiled oligos will typically be too concentrated to pipet directly, so you may need to dilute an aliquot before pipeting the assembled oligos into the ligation.