Real-time Optimisation. A). Real-time amplification plot. Three real-time amplification plots are shown for GAPDH as target 2, GLI as target 1 and no-template controls (background at the bottom of the graph). DNA from the SJSA1 cell line was used for this experiment. The cycle numbers (cycle threshold, Ct) necessary to achieve the given level of fluorescence of 2 (horizontal line), which lies in the log-linear phase of both PCR reactions, were 27.03 and 32.25 for target 1 and 2 respectively (vertical lines). A difference of 32.65-27.03 = 5.62 cycles between the two Ct, means that there was 25.62= 49.2 more of target 1 relative to target 2. This confirms an amplification of the GLI gene in this cell line. B) and C). Dissociation curves for GAPDH (B) and signal TREC (C) PCR reactions. Dissociation curves provide a graphical representation of the PCR product after the amplification process. A single peak in positive samples (graph +) suggests a single size product (GAPDH amplification from PBMC DNA). An absence of peak in the negative control sample (graph -) indicates the lack of primer dimer. Occasionally, primer-dimers were observed in negative control wells, (as shown by the arrow on plot C for signal TREC amplification from PBMC DNA). The melting temperature of each PCR product varies and is dependent on its sequence and size. A melting temperature of 81°C was observed for the GAPDH product (84 bp), which contained 17 Gs, 29 Cs, 17 As and 21 Ts. The signal TREC product (122 bp) had a melting temperature of 87°C and contained 19 Gs, 25 Cs, 33 As and 45 Ts. The occasional primer-dimers (indicated by the arrow on graph C) have a much lower melting temperature. D) Primer concentration optimization. Three sets of primers were designed to amplify 3 target genes (OPA1, MYC-C and GLI). A primer concentration optimization experiment was performed where all PCR conditions were the same apart from the primer concentration. The same PBMC DNA sample was used in all tests. Normalization was performed against GAPDH in this DNA sample using the delta Ct method. The primer concentration used for GAPDH in this assay was 100 nM. MYC-C (black bars) is optimally amplified at the concentrations of 500 nM for both primers with a ratio of 0.99. GLI (grey bars) is similarly detected over concentrations ranging from 100 to 500 nM for both primers however, with an optimal ratio of 0.98 at 300 nM. OPA1 (open bars) is only detected at a primer concentration of 500 nM with a ratio of 0.95. Of note, excess of primer is detrimental to all 3 PCR assay as amplifications yielded less product for the 3 different targets at concentrations of 1000 nM for each primer.