Top10 e coli bacteria

PCR amplicons of the correct size were TA cloned into pCR2.1 (TA Cloning Kit, Invitrogen) following the manufacturer's protocol and incubated overnight at 14°C. The following day, the ligation reaction was transformed into chemically competent TOP 10 E. coli bacteria (Invitrogen) and grown overnight at 37°C on Luria-Bertani broth agar plates containing 100 µg ml -1 carbenicillin, 40 µg ml -1 X-gal, and 100 µM isopropyl-β-D-thiogalactopyranoside (standard blue/white clo ning). Individual white bacterial colonies (5 clones sample -1 ) were inoculated into 5 ml of LB broth containing 100 µg ml -1 carbenicillin, placed at 37°C, and shaken overnight at 225 rpm. Plasmid DNA was purified (Wizard Plus SV Miniprep DNA Purification System, Promega), digested with EcoRI (New England BioLabs), and visualized using agarose gel electrophoresis. Plasmid clones containing the correct-sized insert were sent to a local vendor (2-3 clones sample -1 ) for sequencing (Davis Sequencing). Putative Bd sequences were digitally compared to the GenBank database using the Basic Local Alignment Search Tool (BLAST, NCBI; www.ncbi.nlm. nih.gov/ blast/Blast.cgi). Representative sequences were digitally aligned and compared (Vector NTI, Invitrogen).