(r5) 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? <br>

Overview <br>

   * Direct PCR of culture or isolation of DNA from culture before PCR. <br>
   * PCR using universal primers. <br>
   * Agarose gel to confirm PCR worked correctly. <br>
   * Excise correct bands from gel and purify DNA. <br>
   * Prepare and submit samples for sequencing. <br>

---++ 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>

It is generally preferred to isolate the DNA from your culture before doing the 16s RNA PCR.  This results in a cleaner PCR product and generally a better sequencing result. <br>

Use the invitrogen purelink genomic kit to isolate DNA from your culture.  <br>
After DNA is isolated, use a nanodrop to find the concentration of DNA.  The ideal amount of DNA for PCR is 0.1 ng/ul (final template concentration) so for a 30ul PCR reaction use 3ng of DNA. <br>

The PCR cycle is as follows: <br>
| 94 | 10 min |
then 40 cycles of
| 94 | 30 sec |
| 54 | 30 sec |
| 72 | 1 min |
then final extension of 72 for 5 min <br>

After PCR, the products need to be run on an agarose gel to confirm correct product (around 1kb) and to excise the correct bands from the gel. <br>

A 1.5%  - 2% gel works the best for this. <br>
After cutting the bands out of gel clean using Zymo gel extraction kit or equivalent. <br>
To prepare DNA for sequencing follow the instructions found here:  http://www.icmb.utexas.edu/core/DNA/DNA_Sequencing/FAQ-Sequencing.htm <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: r5 - 2011-07-15 - 18:08:07 - Main.CraigBarnhart