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Oil-Free PCR Using the Hot Top Assembly

original configuration of the RoboCycler unit or the retrofit configuration. Therefore, the addition of the Hot Top Assembly did not interfere with the formation of an 11C gradient across the annealing block. Experiments performed on a nongradient RoboCycler 96 as well as nongradient and gradient RoboCycler 40 temperature cyclers also showed similar, consistent results when either the standard paddle or the Hot Top Assembly was used (data not shown).

Using the Gradient Mode to Optimize Annealing Temperatures

Figure 2A

figure 2B

RoboCycler Gradient 40 and 96 temperature cyclers aid in experimentally determining optimal annealing temperatures by providing a linear temperature range across the gradient, which allows simultaneous testing of up to 8 or 12 different annealing temperatures.2-5 To optimize an annealing temperature, researchers must be able to choose a temperature from a gradient experiment and consistently reproduce results during subsequent experiments with the annealing block in single-temperature mode. PCR experiments were performed to determine if addition of the Hot Top Assembly to a RoboCycler unit alters the capacity for optimizing annealing temperatures. Reactions were set up and run sequentially, in either gradient or single-temperature mode, on a RoboCycler 96 temperature cycler fitted with a Hot Top Assembly and updated software. As before, human genomic DNA served as the template with primers specific for the glucocerebrosidase gene. The gradient was set to maintain a temperature range from 49 to
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