The transformed cells were then plated onto Luria-Bertani (Promega, Australia) agar plates supplemented with kanamycin (Sigma, Australia) and incubated at 37°C overnight. Ninety six of the resulting bacterial colonies per ligation were picked and grown overnight at 37°C on LB agar plates containing kanamycin. Plasmid BAY 11-7082 in vitro DNA was released from bacterial cells by boiling and one microliter was used as the template in PCR with an M13 forward and reverse primers to determine the correct sizes of inserts. The presence and size of inserts was determined by MI-503 electrophoresing the PCR products on a 1% agarose gel. Subsequently positive PCR products were purified, lyophilized
and sent to Macrogen Inc. (Seoul, South Korea) for sequencing using ABI PRISM® BigDye™ and M13F vector-specific primer. Alignment and phylogenetic analysis The 16S rRNA gene clones of the arterial catheters were divided into two groups, i.e., uncolonised ACs and colonised ACs. The 16S rRNA gene sequences obtained were manually proofread, corrected and edited to start and end with the corresponding primer
nucleotide (using reverse complement transform if necessary) using BioEdit . Sequences with incorrect inserts or with ambiguous bases were excluded from further sequence analyses. Selleckchem CAL101 Vector sequences detected by cross match were trimmed off. Trimmed, assembled sequences were then aligned to a core set of sequences using the NAST alignment tool
on the Greengens website (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). All 16S rRNA gene sequences were screened for potential chimeras using BELLEROPHON Cediranib (AZD2171) which was also available on the Greengens website  and sequences flagged as potential chimeras were discarded from further analysis. Sequences were compared to the NCBI GenBank database using the BLAST program. All examined 16S rRNA gene clone sequences and their most similar GenBank sequences which were not available in the Greengenes database at the time of analysis were identified from BLAST searches of sequences retrieved in this study and were then imported into the ARB software package (http://www.arb-home.de) . OTU determination and diversity estimation The Olsen corrected distance matrix was exported from the ARB program and all sequences were grouped into operational taxonomic unit (OTUs) by the furthest-neighbour algorithm Distance-based Operational Taxonomic Unit and Richness (DOTUR). DOTUR assigned sequences accurately to OTUs based on sequence data using values that are less than the cut off level . A cluster with less than 3% substitutions in the phylogenetic tree was usually matched with the same species or relatives in GenBank as confirmed by the RDP Classifier results. In this study, a similar cut off of 97% was defined as an OTU. This same cut off was used for diversity indices and richness estimates that were calculated by DOTUR.