Immobilizing single molecules Sample Clauses

Immobilizing single molecules. Once the sample chamber surface is prepared, the slide surface should be imaged with imaging buffer before immobilizing nucleic acids to the surface. The ‘blank’ surface should have few fluorescent spots (Figure 2.6A) (any background fluorescence on the slide should be distinctly dimmer at the same excitation than surface- immobilized fluorophores). If this is not the case, an experiment should not proceed. The background fluorescence from slide contamination will interfere with the quality of the single molecule measurement, so the source of fluorescent contamination should be identified and corrected. If the slide is sufficiently clean, the slide surface should be tested for non-specific binding. To check for non-specific binding to the surface, donor- labeled DNA can be added to the slide and subsequently washed out, and the slide should be imaged with imaging buffer. No donor-labeled DNA molecules should be on the surface, since streptavidin is not present. After testing the chamber surface for background fluorescence and non-specific binding, specific single molecule immobilization is accomplished by adding streptavidin and then biotinylated, donor- labeled DNA/RNA to the sample chamber (see Appendix A for single molecule immobilization protocol). The surface density of the 25 µm × 50 µm imaging area should be ~200-300 fluorescent molecules (Figure 2.6B).