Finally, 200 μl of Qiagen buffer AL was added. Samples were mixed by pulse-vortexing for 15 sec. From this point onward, purification was carried out as per manufacturer’s instructions. Finally,
the DNA was eluted in 100 μl of AE buffer from the kit. The DNA concentrations in the samples were measured by using the Quant-iT PicoGreen dsDNA assay kit (Molecular Probes, Invitrogen USA) and ranged from 0.33 ng/μl to 1.59 ng/μl. 16S rDNA PCR DNA (10 μl of 1:9 dilution) was amplified by PCR using the broad range 16S rDNA primers described in Table 1. The composite primers each comprised a 17-20 bases target specific region at their 3′ end and a 19 bases region of the Primer A (forward primer) or the Primer B (reverse primer) sequences needed for selleck compound GS FLX amplicon sequencing (454 Life
Sciences, USA) at their 5′end. PCR reactions were performed using 25 μl (final volume) mixtures containing 1× GeneAmp PCR Gold Buffer Applied Biosystems, 3.5 mM MgCl2, 0.2 mM GeneAmp dNTP, 10 pmol of each primer and 0.025 U/μl AmpliTaq Gold DNA Polymerase, LD (Applied Biosystems, USA). The amplification protocol for the V1V2 amplicon primers was: 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 50°C for 30 s and 72°C for 25 s, and a final elongation step at 72°C for 7 min. The protocol for the V6 amplicon primers was: 95°C for 10 min, find more followed by 35 cycles of 95°C for 30 s, 50°C for 25 s and 72°C for 25 s, and a final elongation step at 72°C for 7 min. Replicate PCRs were performed for each sample. A positive
control (with previously amplified bacterial DNA) as template was run for every PCR. Table 1 PCR primers used Primer Sequence (5′→3′) 16S rDNA region Product size Reference A2+V1 F GCCTCCCTCGCGCCATCAGAGAGTTTGATCMTGGCTCAG V1V2 392 bp 3 [32] B2+V2 R GCCTTGCCAGCCCGCTCAGCYNACTGCTGCCTCCCGTAG 8-361 1 A2+1061R GCCTCCCTCGCGCCATCAGCRRCACGAGCTGACGAC V6 316 bp 3 [33] B2 +784F GCCTTGCCAGCCCGCTCAGAGGATTAGATACCCTGGTA 784-1061 1 The table contains primer name, sequence (hypervariable specific sequence in bold font), 16S rDNA region covered, product size and references for the primers used in this study. 1 Coordinates are given relative Phospholipase D1 to the 1542 bp E. coli K12 16S rDNA sequence. 2 A and B primer: corresponds to 454-adaptor sequences from the amplicon pyrosequencing protocol for GS FLX http://www.my454.com/downloads/protocols/Guide_To_Amplicon_Sequencing.pdf[101], p. 7. 3 Product size includes the primer sequences. PCR amplicons were detected and confirmed for DNA from all eight subjects by agarose gel Combretastatin A4 order electrophoresis prior to pyrosequencing (data not shown). All crucial steps during DNA isolation and the entire PCR set up were performed in a laminar air flow (LAF)-bench, illuminated with a UV lamp prior to use in order to avoid possible contaminants. In addition, negative DNA extraction controls (lysis buffer and kit reagents only) were amplified and sequenced as contamination controls.