Remember, only double stranded DNA that has biotin on one of the strands can be denatured. Typically, it will be a PCR product that was amplified with one biotinylated primer and the other must be non-biotinylated. If both strands are biotinylated you cannot easily remove the DNA from the beads without destroying the streptavidin completely.
1. After immobilization of biotinylated PCR DNA, place the 1.5 mL tube on a magnetic rack for 2 min to collect the beads onto the side of the tube.
2. Using a pipette, carefully remove and discard the clarified supernatant taking care not to dislodge the pellet.
3. To denature the non-biotinylated strand, add 200 µl of 100 mM NaOH (freshly prepared from 10N stock) to the beads and vortex to resuspend the beads. Incubate for 60 sec to denature the non-biotinylated strand.
4. Place the microfuge tube back on the magnetic rack for 2 min.
5. Carefully remove and discard the clarified supernatant.
6. Neutralize any residual base by washing the beads 2X with 500 µL of 0.1 M Tris-HCL, 150 mM NaCl.
7. The single stranded DNA immobilized on the beads is now ready for hybridization by addition of denatured complementary target DNA in a suitable hybridization buffer.
Therefore, if you were to use the biotinylated oligo with our streptavidin-coated magnetic beads, you would easily be able to capture the complementary strand of the biotinylated oligo and then remove it with 100 mM NaOH, which would quickly break the hydrogen bonds, leaving the DNA backbone intact. This type of de-hybridization scheme is much easier to buffer exchange than the detergent method.