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Oil-Free PCR Using the Hot Top Assembly

ween 37 and 99C. RoboCycler 40 temperature cyclers that are equipped with Hot Top Assemblies do not need an upgraded version of software to maintain precise temperature control.1

Comparison of PCR Results: Before and After Addition of a Hot Top Assembly

figure 1

Experiments were performed using a RoboCycler Gradient 96 temperature cycler* to determine if replacing the standard paddle with the Hot Top Assembly and installing the software update PC card would alter the yield and/or specificity of PCR products. Human genomic DNA was used as the template in a PCR amplification with primers specific for glucocerebrosidase, the gene associated with human Gauchers disease. An 11oC temperature range was generated across the gradient block, providing annealing temperatures from 49 to 60C. Each well in a row differed from the preceding well by 1C. A PCR master mix was made, and tubes containing 50-l aliquots of the mix were placed into wells that corresponded to each of the 12 different annealing temperatures in the gradient range. Reactions were cycled under the appropriate conditions on a RoboCycler Gradient 96 temperature cycler fitted with the standard paddle and the original software. The standard paddle was then replaced with a Hot Top Assembly, and the software was upgraded using the PC card. A new PCR master mix was made, aliquoted into tubes and cycled as before. After both experiments were complete, equal volumes of the PCR products were electrophoresed through the same gel, stained with ethidium bromide and visualized using the Eagle Eye II still video system (figure 1). Equivalent banding patterns were observed when using either the
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