Socompa Stromatolite 16S pyrotags
Whole DNA was extracted from 0.5 g of  freeze-dried stromatolite was washed in 10 ml of 10% NaCl and incubated at 40°C for  15 min and then centrifuged at 8200 x g for 15 min (three times).  The SN (approx. 15 ml) is maintained for the precipitation of the EPS (a), while the  pellet is retained for the extraction of genomic DNA (b). Precipitation of EPS was  done according to Hong Liu, et al, with some adaptation2: The supernatant is mixed  with 100% ethanol and taken to a concentration of 70% ethanol and incubated O.N  at 4°C. Centrifuged 15 min at 4000 x g at 20°C. The supernatant is discarded and the  resulting pellet was solubilized in sterile H2O and dialyzed against H2Od in a 10.000 Da  MWCO membrane for 3 hours (3 times) at 4°C. Half was lyophilized for dry weight  determination, while the other half was used for chemical analysis and HPLC. Genomic  DNA extraction was done according to Falcon et al1, whit modifications : The cell pellet  of sediment was resuspended in lysis buffer (Tris-HCl, pH 8, 100 mM NaCl 1.5 M,  EDTA, pH 8, 100 mM; Na3PO4, pH 8, 100 M; CTAB 1%). The cells were frozen and  thawed first in N2 (l) and then in a water bath at 65°C (three times). The mixture was  incubated with lysozyme (1 mg / ml) for 30 min at 37°C, then added Proteinase K (0.1  mg / ml), 3% final SDS and incubated ON at 60°C. Supernatant were collected after  10 min of centrifugation at 5000 x g. Organic extractions are made first with 1 Vol.  of phenol:chloroform:isoamyl acohol (25:24:1) and then with 1 Vol. of chloroform:  isoamyl alcohol (24:1). DNA (aqueous phase) is separated and precipitated with cold  Isopropanol (0.7 Vol.) and Sodium Acetate 3M (0,1 Vol), incubated 2 hours at -20°C  and then, the DNA precipitate is washed (three times) with cold 70% ethanol, collecting  the DNA between each wash with centrifugation for 15 min 15000 x g at 4°C. Finally,  the DNA is dried in sterile air and resuspended in DNAse-free H2O.  The V4 hyper variable region of the 16s rRNA gene was amplified using the following  universal primers containing the Roche 454 sequencing A and B adaptors (underlined)  and a 10 nucleotide “multiple identifier” (MID) to sort samples (bold): 520F (5’-  CGTATCGCCTCCCTCGCGCCATCAGAGCACTGTAGAYTGGGYDTAAAGNG-3’,  CTATGCGCCTTGCCAGCCCGCTCAGTACCRGGGTHTCTAATCC,  CTATGCGCCTTGCCAGCCCGCTCAGTACCAGAGTATCTAATTC,  and CTATGCGCCTTGCCAGCCCGCTCAGCTACDSRGGTMTCTAATC,  CTATGCGCCTTGCCAGCCCGCTCAGTACNVGGGTATCTAATCC) obtained from  RDP’s Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help. Five independent  PCRs were performed. The PCR mix (final volume 25 ul) contained 2.5 ulFastStart  High Fidelity 10X Reaction Buffer (Roche Applied Science, Mannheim, Germany),  20 ng of template DNA, 0.4 uM each primer, 1.25 U FastStart High Fidelity Enzyme  Blend (Roche Applied Science), and 0.2 mM DNTPs. The PCR conditions were 95 °C  for 5 minutes for initial denaturalization, followed by 95 °C for 45 seconds, 57 °C for  45 seconds, 72 °C for 60 seconds in 30 cycles, and a final elongation step at 72°C for  4 minutes. Two negative control reactions containing all components were performed  without the template. The five reactions were pooled and purified using AMPure  beads XP. Quantification of the purified PCR product was performed by using Quant-  iTPicoGreendsDNA Kit (Invitrogen Molecular Probes Inc, Oregon, USA).  Purified PCR product were immobilized onto DNA capture beads, amplified  through emulsion-based clonal amplification (emPCR), and sequenced together  in a PicoTiterPlate device on a Genome Sequencer FLX (Roche Applied Science)  using Titanium Chemistry according to the manufacturer’s instructions at INDEAR  (Argentina) Genome sequencing facility.
INDEAR
Run 13 Socompa Stromatolite 3