( eratures facilitating room-temperature...)Greater Amplification Specificity with New Hot Start PCR Enzyme

eratures, facilitating room-temperature PCR setup and high-throughput PCR analyses. Moreover, high sensitivity and room-temperature assembly can be achieved with little or no changes to existing procedures. The benefits of SureStart Taq DNA polymerase in quantitative PCR and RT-PCR applications were highlighted in a previous article that discussed Stratagenes new Brilliant PCR and RT-PCR core reagent kits.¹²

Methods

PCR reactions were assembled at room temperature. PCR amplifications (50 ml) employed 200 mM each dNTP, 0.3 to 0.5 mM each primer, and the indicated amounts of DNA template (figure legends). Amplifications were carried out using 2.5 U of SureStart Taq or Taq2000 DNA polymerase (Stratagene) and the recommended PCR buffer. Enzyme comparisons with other vendors hot start Taq antibody or hot start Taq preparations were carried out under identical conditions, except that the recommended PCR buffer and activation method was used.

PCR reactions were amplified in a RoboCycler Gradient 96 temperature cycler (Stratagene) fitted with a Hot Top device and using 200-ml thin-walled PCR tubes. Enzyme comparisons employed identical temperature cycling parameters, except that a heat step consisting of 93C to 95C for 10 minutes was added to the beginning of each thermal cycling program for SureStart Taq reactions. The 105-bp target (Figure 1) was amplified using the following conditions: 1 cycle at 95C for 1 minute (Taq) or 10 minutes (SureStart Taq), followed by 30 to 42 cycles at 95C for 1 minute (denaturation); 54C for 1 minute (annealing); and 72C for 1 minute. The conditions used to a
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