Spiked samples were subjected to DNA-extraction and real-time PCR as described above. Community PCR Template DNA obtained from cheetahs B1 and B2 was subjected to 16S rRNA gene amplification using the conserved primers pA (5′ AGA GTT TGA TCC TGG CTC AG 3′) and pH (5′ AAG GAG GTG ATC CAG CCG CA 3′) which flank respectively the extreme 5′ and 3′ part of the 16S rRNA gene, thus allowing amplification of the entire gene [25]. Each reaction mixture (50 μl) contained 5 μl 10x PCR buffer (100 mM Tris–HCl, pH 8.3 [at 25°C]; 500 mM KCl; 15 mM MgCl2; 0.01% [wt/vol] gelatin [GeneAmp®; Applied Biosystems, USA]), 1 μl 25 mM MgCl2, 5 μl 2 mM dNTPs (GeneAmp®;
Applied Biosystems, USA), 0.04 μl 10 μg/μl bovine serum albumin, 1.25 μl 1 U/μl AmpliTaq® (Applied Biosystems, USA), 2.5 μl of each 10 μM primer, 4 μl template DNA and milliQ water to 50 μl. The samples Staurosporine nmr were amplified in the Veriti™ Dx 96-Well Thermal Cycler (Applied Biosystems, Selleckchem BIBW2992 USA), using the following PCR programme: initial denaturation at 94°C for 5 min followed by 18 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for
1 min, with a final extension of 72°C for 10 min. Negative (milliQ water as template) and positive controls (Marinobacter sp. strain T278 [R-39409]) were included in parallel. Amplicons were checked on a 1% agarose gel under UV illumination after ethidium bromide staining of the gel, and subsequently purified with the QIAquick® PCR purification kit (Qiagen, Germany). Cloning of bacterial 16S rRNA gene amplicons For both cheetahs B1 and B2, a clone library was prepared. Purified 16S rRNA gene amplicons
were ligated into the pGEM®-T Vector System (Promega Benelux, The Netherlands) and transformed into Phosphatidylinositol diacylglycerol-lyase competent E. coli cells according to the manufacturer’s instructions. White clones were amplified using the primer pair T7 (5′ AAT ACG ACT CAC TAT AGG 3′) and Sp6 (5′ ATT TAG GTG ACA CTA TAG 3′) to determine the size of the inserts. Sequencing and sequence processing The diversity of the clone libraries was examined via short fragment sequencing on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA) by means of the Big Dye® XTerminator™ v.3.1. Cycle Sequencing and Purification Kit (Applied Biosystems, USA) according to the protocol of the supplier. The sequencing primer used was BKL1 [26]. For each sample, clones were sequenced, assembled in BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium) and edited to exclude the primer binding sites. Chimeras were detected using Bellerophon [27] and B2C2 [28], and excluded for further analysis. find more Phylogenetic analyses Chimera-free sequences were aligned using ClustalW in MEGA 5.0 [29] and corrected by manual inspection. Homology searches were performed via BLAST [30], and taxonomic classification of the 16S rRNA transcripts was obtained by comparison against The Ribosomal Database Project-II (RDP) [31]. Only annotations with a bootstrap value over 0.