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 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: r4 - 07 Jun 2011 - 16:25:17 - Main.CraigBarnhart
 
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