Supporting information for Maheshri and Schaffer (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0537968100
Fig. 6.
Thermal melt curve analysis. An ≈100-bp portion of the cytomegalovirus (CMV) promoter amplified by PCR was subjected to a melt curve analysis, using the Bio-Rad iCycler. The DNA was equilibrated at 95ºC for 6 min with reaction conditions identical to those used for the GFP shuffling except for the addition of SYBR green and 10 nM fluorescein dye (for calibration purposes). The temperature was then ramped down in 0.5ºC increments, with either a 5- or 30-sec pause at each step. Relative fluorescent units (RFUs) were monitored by the reaction, and their negative derivative with respect to temperature are plotted on the y-axis. The recommended 30-sec pause shows a peak at ≈89ºC, the true Tm at these conditions, whereas only a 5-sec pause results in annealing occurring at temperatures less than the true Tm, because of kinetic limitations.