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Oil-Free PCR Using the Hot Top Assembly

60C, and the experiment in single-temperature mode was performed with an annealing temperature of 54C. For the single-temperature portion of the experiment, 10 aliquots from a large master mix were positioned throughout the block (figure 2B) in order to test well-to-well consistency. Following the gradient and single-temperature runs, aliquots of PCR products were electrophoresed through the same gel, stained with ethidium bromide and visualized using the Eagle Eye II still video system (figure 2A). Banding patterns from the 54C single-temperature annealing experiment closely matched those observed for the corresponding temperature tested within the gradient range, indicating that the Hot Top Assembly did not diminish the ability to optimize annealing temperatures using the gradient block. In addition, the Hot Top Assembly did not alter the well-to-well consistency when using the gradient block in uniform mode.

Cycle Sequencing Using the Hot Top Assembly

Unlike conventional PCR experiments where reaction volumes of 25 to 100 l are common, cycle sequencing experiments are performed using very small reaction volumes. Consequently, cycle sequencing is likely to be sensitive to small amounts of liquid lost from reactions during cycling because this amount makes up a larger percentage of the original reaction volume than in a standard PCR experiment. Cycle sequencing also requires small volumes of radioactive liquid under oil overlays to be removed from tubes for loading onto a sequencing gel. For these reasons, cycle sequencing is a good experimental test of the ability of the Hot Top Assembly to decrease condensation formed during cycling and to preserve the quality of results obtained using the standard paddle.

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