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).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/