Jm109 escherichia coli cells

All target genes (Table 2) were amplified by PCR with a Primus Thermo cycler (MWG Biotech, Ebersberg, Germany) under the following conditions: 95°C for 4 min, 35 cycles (94°C for 30 s, annealing for 30 s at 60°C, 72°C for 30 s), and a final 10 min extension at 72°C. After confirmation by 1.5% agarose gel electrophoresis, 3 μL PCR products were purified on spin columns according to the manufacturer’s protocol (Roche, Mannheim, Germany), cloned into the pGEM-T Easy vector according to the manufacturer’s protocol (Promega, Mannheim, Germany) and transformed into competent JM109 Escherichia coli cells (Promega). Sequencing was conducted by Eurofins MWG Operon (Ebersberg, Germany).

To assess the microbial diversity within leech shed mucus, total DNA was extracted from 3 d old mucosal sheds of two individuals, using the Holmes-Bonner protocol (Holmes and Bonner, 1973 (link)), and subjected to PCR using 27F′ and 1492R′ general eubacteria primers (Lane, 1990 ; Weisburg et al., 1991 (link)) (T_a [annealing temperature] = 50°C; 28 cycles; amplicon ~1450 bp). PCR products were cloned using the pGEM-T Easy Vector cloning kit (Promega, WI), with subsequent transformation into JM109 Escherichia coli cells (Promega, WI). Inserts were amplified using M13F′ and M13R′ vector primers (T_a = 46°C; 35 cycles; amplicon ~1636 bp) and digested with HaeIII restriction endonuclease (NEB, Ipswich, MA, USA) for RFLP typing. Clones with unique restriction profiles were purified and subject to Sanger sequencing using M13 primers with an ABI Genetic Analyzer 3130xl at the WVU Department of Biology Genomics Center. The DNA sequences were aligned and assembled into contigs and identified to the highest taxonomic level possible using nucleotide Basic Local Alignment Search Tool (BLASTn, http://blast.ncbi.nlm.nih.gov/Blast.cgi).