This protocol is aimed at amplifying the 16S rRNA hypervariable region 4 (16S V4) in prokaryotes. The primers (515fB and 806rB, 926R) used in this protocol are based on the primer utilized in Apprill et al 2015 and the Earth Microbiome Project (EMP).This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.Citation Apprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 75, 129–137 (2015). doi:10.3354/ame01753Earth Microbiom Project http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/