Figure 1
Primer optimization and visual verification of a single amplicon for maize chloroplast genes utilizing endpoint RT-PCR. Lanes 1 through 8 denote an annealing temperature gradient, Panel A shows amplification of psbB mRNA and represents a typical primer optimization run for a Category 1 primer set. In addition to temperature, psbB's optimization elongation time was 2 minutes. Reducing the elongation time to 30 seconds yielded equivalent results. Panels B and C show results for psaB primer pairs. Panel B shows multiple products due to a 2 minute extension time and 5' and 3' primers with a 4°C difference in annealing temperatures. Panel C shows end-point PCR results with a 30 second extension time and the 5' and 3' primers re-designed to have melting temperatures with less than 1°C difference. Panel D represents the optimized annealing temperatures for a single amplicon of rpl14 indicative of category 2a. Panel E represents the optimized annealing temperatures for an atpI amplicon that is indicative of category 2b. Items F and G illustrate q2(RT)PCR amplification (F) and melt curve (G) of a category 1 primer set for matK optimized at different annealing temperatures. In Panel F the x-axis denotes cycle number, the y-axis denotes the base-line subtracted recorded fluorescence units and demonstrates that overall efficiency of the reactions deviate slightly but the crossover thresholds remain consistent. In Panel G the x-axis denotes temperature, the y-axis denotes the change in recorded fluorescence units, and confirms all annealing temperatures produce a single amplicon with a melting temperature of 76.00°C. Panel A annealing temperatures 1 = 59.0°, 2 = 58.2°,3 = 57.0°, 4 = 55.1°, 5 = 52.7°, 6 = 50.9°, 7 = 49.7°, 8 = 49.0°. Panel B 1 = 63.0°, 2 = 62.0°, 3 = 60.1°, 4 = 57.2°, 5 = 53.2°, 6 = 50.4°, 7 = 48.4°, 8 = 47.2°. Panel C 1 = 60.0°, 2 = 59.3°, 3 = 58.2°, 4 = 56.4°, 5 = 54.3°, 6 = 52.7°, 7 = 51.6°, 8 = 51.0°. Panel D 1 = 62.0°, 2 = 60.9°, 3 = 59.0°, 4 = 56.1°, 5 = 52.5°, 6 = 49.9°, 7 = 48.0°, 8 = 47.0°. Panel E 1 = 64.0°, 2 = 63.1°, 3 = 61.6°, 4 = 59.3°, 5 = 56.4°, 6 = 54.3°, 7 = 52.8°, 8 = 52.0°.