How can I check the efficiency of my reaction to create duplexes?

The annealing efficiency of oligos to be duplexed can be checked on a non-denaturing acrylamide gel. For oligos ordered separately (two different tubes) the end-user can run three lanes: one lane for the "duplexed" oligos, and one lane for each of their single-stranded oligos. The ssDNA will migrate faster than the dsDNA (the smaller size allows for faster gel migration than the larger negative charge of the duplex). Single-stranded oligos cannot be visualized with Ethidium Bromide (this works for only double-stranded oligos), instead the end-user must use a visualizing reagent that can detect ssDNA or ssRNA ("Gel-Star" is a reagent that can be used for this).

Once properly visualized the dsDNA fragment can potentially be cut from the gel, thus purifying the double-stranded fragment.

General conditions are as follows:

  • use 15% Non-denaturing Acrylamide gel
  • use Gel-star to visualize the oligos
  • run dsDNA and ssDNA in separate lanes (single-stranded oligos will run faster/migrate further)

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