Brucella DNA was prepared as previously described [27 (link)]. PCR amplification was performed in a total volume of 15 μl containing 1ng of DNA, 1× PCR Reaction Buffer, 1 U of Taq DNA polymerase (Qbiogen, Illkirch, France), 200 μM of each deoxynucleotide triphosphate, and 0.3 μM of each flanking primer as described previously [15 (link)].
Amplifications were performed in a MJ Research PTC200 thermocycler. An initial denaturation step at 96°C for 5 minutes was followed by 30 cycles of denaturation at 96°C for 30 s, primer annealing at 60°C for 30 s, and elongation at 70°C for 1 min. The final extension step was performed at 70°C for 5 min.
Two to five microliters of the amplification product were loaded on a 3% standard agarose gel for analyzing tandem repeats with a unit length shorter than 10 bp and on a 2% standard agarose gel for all others, and run under a voltage of 8 V/cm until the bromophenol blue dye had reached the 20 cm position. Gels were stained with ethidium bromide, visualized under UV light, and photographed (Vilber Lourmat, Marnes-la-Vallée, France). A 100-bp and a 20-bp ladder (EZ Load 100 pb or 20 bp PCR Molecular Ruler, Biorad, Marnes-la-Coquette, France) were used as molecular size markers depending on the tandem repeat unit length. Gel images were managed using the Bionumerics software package (version 4.0, Applied-Maths, Belgium).
Amplifications were performed in a MJ Research PTC200 thermocycler. An initial denaturation step at 96°C for 5 minutes was followed by 30 cycles of denaturation at 96°C for 30 s, primer annealing at 60°C for 30 s, and elongation at 70°C for 1 min. The final extension step was performed at 70°C for 5 min.
Two to five microliters of the amplification product were loaded on a 3% standard agarose gel for analyzing tandem repeats with a unit length shorter than 10 bp and on a 2% standard agarose gel for all others, and run under a voltage of 8 V/cm until the bromophenol blue dye had reached the 20 cm position. Gels were stained with ethidium bromide, visualized under UV light, and photographed (Vilber Lourmat, Marnes-la-Vallée, France). A 100-bp and a 20-bp ladder (EZ Load 100 pb or 20 bp PCR Molecular Ruler, Biorad, Marnes-la-Coquette, France) were used as molecular size markers depending on the tandem repeat unit length. Gel images were managed using the Bionumerics software package (version 4.0, Applied-Maths, Belgium).
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