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 20 µL.
- Boil in water (~500-600 mL volume) for 10 min. Turn off flame
and allow to cool slowly on bench
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
2015 Department of Biology, Davidson College, Davidson, NC 28036
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