This application note describes the experimental steps for measuring the hybridization between a quencher (DABCYL) labeled PNA probes and a fluorescein labeled DNA using the Fluorescence BioMelt Package
The document details an experiment measuring the thermal stability of hybridization between DABCYL-labelled PNA probes and 5'-6-carboxyfluorescein-labelled DNA. It compares the thermal melt (Tm) values of 9mer and 13mer PNA probes bound to the same DNA species.
The package automatically performs thermodynamic calculations such as hybridization enthalpy, entropy, free energy, and rate constants to establish optimal experimental conditions.
The samples were prepared at 50 nM in a volume of 1 mL buffer solution containing 100 mM NaCl, 10 mM KPO4 at pH 7.1.
The thermal melt value for the 13mer probe was 63.9 °C, while the value for the 9mer probe was 49.94 °C.