M. vertebralsi microbiome
"Eubacterial 16S rRNA genes were PCR amplified from extracted genomic DNA [PCR in 50 ?L containing 20 ng DNA, molecular biology grade water, 1X PCR Buffer (Qiagen), 200 ?M of each of the dNTPs (Invitrogen), 1 U HotStarTaq DNA Polymerase (Qiagen), 25 pmoles each of the primers 63F (5`-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNCAGGCCTAACACATGCAAGTC) and 533R (5`-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTTACCGCGGCTGCTGGCAC) modified on the 5? end to contain the 454 FLX adapters A and B, respectively]. The forward primers also contained an eight base barcode sequence positioned between the primer sequence and the adapter. A unique barcode was used for each sample. Thermocycling conditions were as follows: 95?C for 5 min; then 30 cycles of 94?C for 1 min, 55?C for 1 min, 72?C for 1 min; then 72?C for 10 min. Five amplicons were performed per sample and then pooled to give a final quantity of ~1 ?g DNA per sample. Amplicons were purified using a MO BIO PCR purification kit as per the manufacturer?s instructions. The amount of PCR amplified DNA is each sample was quantified using the Quant-iT PicoGreen assay (Invitrogen, Carlsbad, CA). All samples with their respective bar codes (46 samples in total) were pooled in eqimolar amounts for 454 pyrosequencing on a Roche GS-FLX system at the Australian Genome Research Facility (AGRF) Brisbane, Australia."