FRET detection method
To monitor the ligation reaction in online kinetics and microfluidics a FRET-quenching system was developed, containing a Dabcyl-modified Ellman-derivative of Ellman's reagent as a quencher leaving group.
Preliminary experiments revealed a fast ligation reaction allowing replication experiments even at submicromolar concentrations in a reasonable time scale. This led us to choose 9mer educts for ligation to provide a sufficient population of the termolecular complex. We designed two 9mer-18mer complementary system with a dabcyl labelled leaving group and an Alexa488 fluorescent dye label in one precursor building block in 3'- and 5'- position, respectively.
Figure 30