Bacterial diversity and community composition from seasurface to subseafloor in the Equatorial Pacific analyzed by pyrotag sequencing: Sample EQP_1WC24
Construction protocol: The v6-v4 hypervariable regions of the SSU rRNA gene were amplified using degenerate primers. The primers were1064R (CGACRRCCATGCANCACCT) and 518F (CCAGCAGCYGCGGTAAN) fused to Roche GSFLX amplicon sequencing adapters and including 5 nt multiplexing barcodes. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 1.0 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 uM of each primer. We added approximately 10-25 ng template DNA to each PCR and ran a no-template control for each primer pair. Amplification conditions were: initial 94C, 3 minute denaturation step; 30 cycles of 94C for 30s, 60C for 60s, and 72C for 90s; final 10 minute extension at 72C. The triplicate PCR reactions were pooled after amplification and purified using Ampure beads. Purified DNA was eluted in 30 uL of Qiagen buffer EB. We assessed the quality, size and concentration of PCR products on a Perkin Elmer Caliper GX.  Reads were demultiplexed and barcodes removed for submission.
MBL
EQP_1WC24_Bv6v4