3. Methods

3.1 Genomic DNA Isolation

Genomic DNA of sufficient purity for PCR amplification can be isolated using the corresponding kits from Mo Bio (see Materials and Notes 1 and 2). Alternatively, if there are difficulties in obtaining these kits, total genomic DNA can be isolated using the following protocol:

1. To 200 mg of material (microbial or cell culture pellet, soil, plant tissue, or other sample), in a 2-mL screw-top cryogen storage propylene plastic tube, add 0.1 g zirconium beads (0.1-mm diameter), 700 ^L lysis solution (50 mM Tris-HCl, 10 mM EDTA, 2% sodium dodecyl sulfate [SDS], pH 8.0), and 700 ^L Tris-buffered phenol, pH 8.0.

2. Shake the tubes ("homogenize" setting on a Mini Bead-Beater-8™ [Biospec Products, Bartlesville, OK]) for 1 min and chill on ice for 2 min.

3. Repeat the previous step twice.

4. Centrifuge the tubes in a tabletop centrifuge at 10,000g for 5 min.

5. Transfer the upper water phase to a 2-mL microcentrifuge tube and extract with phenol-chloroform-isoamyl alcohol (25:24:1) twice or until the interphase is clear.

6. Precipitate with ethanol, wash with 70% ethanol, dry, and dissolve in 50 ^L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 10 ^g/mL RNaseA.

3.2. Conventional PCR Detection of RPP Genes

1. On ice, combine the following components of the PCR mixture in a 200-^L thin-walled PCR tube: 25 pmol of each primer, 1X ExTaq reaction buffer, 100 ^M of each

Table 2

PCR Primers Targeting tet Efflux Pumps of Gram-Negative Bacteria

Table 2

PCR Primers Targeting tet Efflux Pumps of Gram-Negative Bacteria

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