(r4) Protocols16SSequencing < Lab

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---+ 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. <br>

---++ 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).

Barrick Lab > ProtocolList > Protocols16SSequencing

Topic revision: r4 - 2011-06-07 - 16:25:17 - Main.CraigBarnhart