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)