Figure 3

Fluorometric melting curve analysis of complimentary and mismatched templates to determine optimal Tm and annealing rate. (A) Full complimentary; (B) reverse single mismatch; (C) 1 × 1 oligonucleotides; (D) 2 × 1 oligonucleotides. Average negative first derivative calculations of normalised fluorescence data was plotted to visualise melting temperature (solid line). Average annealing rate was calculated by measuring the gradient of the logarithmic transformed curve of the normalised fluorescence data across the calculated Tm ± 1°C, and plotted across 5°C intervals spanning 20-80°C (dashed line). Data for both Tm and annealing plots is represented as the mean of three replicates from a single experiment performed, which was typical of three repeated experiments.