Building dsDNA with Oligos
- Prepare 10X annealing buffer (1M NaCl; 100mM Tris-HCl,
pH 7.4). This will be done for you.
- Design oligos using the "Oligator".
You do this in lab.
- Synthesize DNA oligos (a company will do this for you).
Store -20° C. We prefer to have the
company produce them at 100 µM.
- Mix all the oligos and buffer (1X final concentration)
for a total volume of 20 µL. Use 0.5 mL microfuge tube.
- 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
- 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.
- If sticky ends were designed, go directly to ligation.
Otherwise, digest and proceed accordingly.
- You could directly to ligation
and use the normal ligation formula to
determine how many nanograms you want for your ligation.
- 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 µL of the assembled oligos into the
2015 Department of Biology, Davidson College, Davidson, NC 28036
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