FIG. 3.

PCR analysis of RD₂₃. Primers for the RD₂₃ PCR assays were positioned an additional 1 to 2 kb from the left and right ends of the respective RD to ensure inclusion of the junctions in the amplicons. PCRs were performed with 25-μl volumes containing 5 mM MgCl₂ and 160 μM of each deoxynucleoside triphosphate (Idaho Technology, Salt Lake, UT), 500 nM (each) of forward and reverse primer, and 2.5 U of Platinum Taq (Invitrogen). Thermocycling conditions were optimized and performed using both T-Gradient (Biometra, Göttingen, Germany) and Dyad (MJ Research, Reno, Nevada) thermocyclers according to the following cycling parameters: initial hold at 95°C for 2 min, 30 s; 30 cycles of 95°C for 30 s, 64°C for 1 min, and 72°C for 1 min; final extension at 72°C; and a final indefinite hold at 4°C. PCR products were electrophoresed on an agarose gel and stained with ethidium bromide. Strain designations are indicated above the respective lanes. The marker lane on the left contains the 1-kb ladder with sizes of the 1- to 3-kb fragments indicated to the left. (For more information on strain designations, see Table S1 in the supplemental material.)