<noautolink> ---+ 16S rRNA Sequencing to Identify Unknown Microbes Ever wonder what that contaminant in your culture is? Need to accurately identify an environmental isolate? ---++ PCR Reaction For PCR we use universal primers U341F and UA1406R that amplify an approximately 1 kb stretch of rDNA and should work for nearly any bacterial or archaeal sequence [1]. | *primer* | *sequence* | | U341F | CCTACGGGRSGCAGCAG | | UA1406R | ACGGGCGGTGWGTRCAA | The dilution of cells used in the reaction plays a critical role in the success of the amplification. Too many cells or components of certain media can inhibit PCR. For best results take a visible turbid overnight culture from a rich medium and dilute approximately 10,000-fold into the final PCR. Ideally, make a dilution series of the template cells. The optimal dilution is somewhere between 1000 and 10,000 fold. PCR setup should include an initial denaturing step: 10min at 94 C. ---++ Analysis Use the tools at the [[http://rdp.cme.msu.edu/][Ribosomal Protein Database]]. ---++ References 1. Baker, G.C., Smith, J.J. & Cowan, D.A. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55, 541-55 (2003).
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Topic revision: r3 - 2011-04-25 - CraigBarnhart