Clone library preparation from community DNA
Total community DNA was used for preparing 16S rRNA gene libraries. The 16S rRNA gene was amplified with modified universal primers for bacteria 8FI (5’GGATCCAGACTTTGATYMTGGCTCAI-3’) and 907RI (5’- CCGTCAATTCMTTTGAGTTI-3’) CUDC-907 solubility dmso [27]. The PCR product were purified by gel elution using Gene Elute Gel Extraction Kit (Sigma-aldrich, St Louis USA) and were ligated into pCR4® TOPO vector supplied with the TOPO TA cloning kit (Invitrogen, San Diego, USA) and transformed into One Shot TOPO10 electrocompetent cells of E. coli (Invitrogen, San Diego, USA) following the manufacturer’s instructions. Sterile LB agar with 50 μg/ml of kanamycin were used for selection of the transformed cells which were incubated for 16 h at 37°C. M13F and M13R primers were used for screening and sequencing of the clones. The sequencing was done by ABI 3730 XL DNA analyser (Applied Biosystems Inc, USA) using the ABI Big-Dye terminator version 3.1 sequencing kit as per the manufacturer’s this website instructions. Phylogenetic analysis Sequences from each of the clone libraries
were compared to the current database of 16S RNA gene sequences at Ribosomal Database Project II [28]. The sequences were assembled and contig’s were obtained using ChromasPro software, alignment was done using CLUSTAL X2 and the sequences were edited manually using DAMBE to get unambiguous sequence alignment. All sequences were checked for chimeric artifacts by Mallard program, reference sequence used for this purpose was E. coli U000096 [29] Appropriate subsets of 16S rRNA gene sequences were selected on the basis of initial results and subjected Pregnenolone to further phylogenetic analysis using DNADIST of Phylip (version 3.61). The number of Operational Taxonomic Units (OTU) (clone sequences with > 97% similarity grouped together as one OTU) were obtained
by DOTUR program (version 1.53) using furthest neighbor algorithm [30]. Representative sequences from each of the OTUs were retrieved and checked against the previously determined 16S rRNA gene from the RDPII release 10 version of the database and these sequences were downloaded in FASTA format. Phylogenetic analyses were conducted using MEGA, version 4 [31], and the phylogenetic trees were constructed using neighbor-joining method with Kimura 2 parameter [32, 33]. Normalized heat map was generated using MG-RAST, a modified version of RAST server, using RDP database [34]. Real time PCR The Real Time PCR was done using the 7300 Real time PCR system from Applied Biosystems Inc. (USA) using SYBR green master mix (Applied Biosystems Inc. USA). Primers used for absolute quantification were reported earlier [19]. The primers used are listed in Table 1.