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Unique DNA Polymerase Formulation Excels in a Broad Range of PCR,,,Applications

NA polymerase High Fidelity (Life Technologies, Inc.), Advantage HF (ClonTech), LA Taq (Takara), and Expand 20 kbPlus PCR system (Roche Molecular Biochemicals). For fidelity assays, PCR amplifications were carried out in each enzymes recommended PCR buffer using 2.5 U of each DNA polymerase.

For PCR comparisons, amplifications (50 l) employed 200 M (<1 kb) or 500 M (>17 kb) each dNTP and 100 ng (<1 kb) or 200 ng (>17 kb) of each primer. The following amounts of DNA template were used: 100 ng (<1-kb targets) or 240 ng (17-kb and 30-kb targets) of genomic DNA or 15 ng (48 kb) of lambda DNA. As recommended by each enzyme manufacturer, amplifications were carried out with 2.5 U (<1 kb) or 5 U (>17 kb) of the Herculase formulation, 2.5 U of Platinum Taq DNA polymerase High Fidelity, or 2.6 U of the Expand 20 kbPlus blend. The following amounts of DMSO were added to Herculase PCRs: 0% (0.1-kb to 17-kb targets), 3% (30-kb target), or 7% (48-kb target).

Reactions were cycled in 200-l thin-walled PCR tubes using a RoboCycler gradient 96 temperature cycler (Stratagene)(<1-kb targets) or a single-block thermocycler (>17-kb targets) fitted with a Hot Top Assembly. For enzyme comparisons, identical temperature cycling parameters were employed that consisted of (<1-kb targets) 1 cycle at 94C for 1 minutes, followed by 30 cycles at 94C for 40 seconds (denaturation), 58C for 40 seconds (annealing) and 68C for 1 minute, or (>17-kb targets) 1 cycle at 92C for 2 minutes, followed by 10 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target, followed by 20 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target plus an additional 10 seconds/cycle. After 30 cycles, one final extension cycle of 68C for 10 minutes was carried out.

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