Labeling DNA (or RNA) for single-molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique for measuring distances in the 30-80 Å range in which excitation energy of a donor molecule is transferred to an acceptor via interaction between two induced dipoles. A variant of smFRET is based on total internal reflection (TIR) microscopy. This protocol describes the labeling of nucleic acids (DNA or RNA) for smFRET with TIR microscopy. The protocol is optimized for 5 nanomoles of DNA that has an amine-modified thymine in its sequence. It is a simplified version of a protocol from Invitrogen.