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Effects of Thermal Cycler Well-To-Well Temperature Uniformity on PCR Results

erature profile for the MJ Research PTC-100 shows that the sample temperature in tubes in the center of the block is up to 1.1C higher than the sample temperature in tubes positioned in the edges of the block (Figure 2). The fluid temperature during the denaturation step varied from 95.3 to 96.4C, with center wells 1.2 to 1.4C higher than the set temperature. The annealing temperature varied from 54.4 to 54.8C, and the extension temperature varied from 72.4 to 73.2C. On average, the PTC-100 changed fluid temperatures at a rate of approximately 0.67C/second, with a coefficient of variation (the standard deviation divided by the mean) of 1.12% between tubes. Therefore, samples placed in different well positions are not only exposed to different temperatures once ramping is complete but also change temperature at different rates. Both of these components combined provide vastly dissimilar PCR conditions between wells of the Peltier-controlled PTC-100.

Figure 2

The temperature profile of the RoboCycler 96 temperature cycler shows a much higher degree of well-to-well temperature uniformity (Figure 2). The fluid temperature during the denaturation step only varied from 95.0 to 95.4C; the annealing temperature varied from 54.4 to 54.6C, and the extension temperature varied from 72.1 to 72.3C. On average, the fluid temperature rate of change for the RoboCycler system was approximately 0.85C/second with a 0.22% coefficient of variation between tubes. This translates to a 27% increase in the average rate of fluid temperature change compared to the PTC-100. Also, unlike the PTC-100, the ramping rates were more consistent from well to well for
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