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deoxynucleoside triphosphate, 1.0 U of ExTaq DNA polymerase, and 200 ng of the purified DNA template, adjusted to a total volume of 20 ^L.

2. Perform PCR amplification (25 cycles) with a GeneAmp PCR system 2400 (Perkin-Elmer, Norwalk, CT) or a DNA Engine Thermocycler (MJ Research, Waltham, MA) using initial denaturation at 94°C for 5 min, followed by cycling at 94° for 30 s, 30 s of annealing (annealing temperatures are shown in Table 2), 30 s of extension at 72°C, with final extension at 72°C for 7 min. Include positive (Table 1) and negative (e.g., a noncomplementary template) controls for each pair of primers used.

3. A second, nested PCR can be performed using 1 ^L of product from the first PCR as a template and amplifying for 25 cycles as described above if PCR amplification fails owing to the presence of unidentified PCR-inhibiting substances.

3.3. Conventional PCR Detection of TEPGNB Genes

1. On ice, combine the components of the following PCR mixture in a 200-^L PCR tube: 25 pmol of each primer, 1X ExTaq reaction buffer, 100 ^M of each deoxynucleoside triphosphate, 1.0 U of ExTaq DNA polymerase, and 125 ng of the purified DNA template, adjusted to a total volume of 25 ^L.

Table 3

Control Strains and Plasmids

Strains and plasmids tet gene Reference/source

RPP genes

Clostridium perfringens JIR4202

tetB(P)

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