HLGB_63_2008Arch
After centrifugation of the mat/sediment/water slurry sample at 3,900*g for 20 min, approximately 5 g was used for extraction via a protocol modified from Zhou et al. (1996; PMID 8593035) that has been described previously (De Le*n et al., 2012; PMID 22476815).    Archaeal small subunit (16S) rRNA primers 751F (5′-CCGACGGTGAGRGRYGAA-3′) and 1204R (5′-TTMGGGGCATRCNKACCT-3′) (Baker et al., 2003; PMID 14607398) were used to amplify the archaeal population with the following PCR protocol:  94*C for 5 min;  cycling 94*C for 30 sec, 55*C for 30 sec, and 72*C for 2 min; followed by 72*C for 7 min and 4*C hold.    All primers had 10-nt barcodes on the 5′ end for sample identification purposes.  Cycle numbers were optimized for each sample to minimize PCR-induced artifacts with 10-12 ng DNA per reaction  Amplicons were sequenced on a 454 GS-FLX Titanium Pyrosequencer at SeqWright, Incorporated (Houston, Texas, USA).