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 genomic DNA from your culture before doing the 16s RNA PCR (using the Invitrogen PureLink kit, for example). This results in a cleaner PCR product and generally a better sequencing result. <br>

After DNA is isolated, use a Qubit to find the concentration of DNA.  The desired amount of gDNA template for PCR is 0.1 ng/ul (final template concentration) so for a 30 ul PCR reaction use 3 ng 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). A 1.5% or 2% gel works the best for this.

If the bands are clean, then you can do a PCR cleanup spin column and submit the DNA for two different sequencing reactions (one with each of your PCR primers). If the bands are not clean, you may be able to excise the band and

The bands should be clean to excise the correct bands from the gel. <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 one of these tools to assign your species:
   * [[http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp][SeqMatch tool]] from the [[http://rdp.cme.msu.edu/][Ribosomal Protein Database]] (preferred)
      * SeqMatch results for <i>Pseudomonas syringae</i>: <br />
     <img src="%ATTACHURLPATH%/Ribosomal_Database_Project.JPG" alt="Ribosomal_Database_Project.JPG" width="853" height="427" />   
   * [[http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=TargLociBlast][Targeted Loci BLAST]] - select the appropriate database for your samples
      * BLAST result for <i>Pseudomonas syringae</i>: <br />
     <img src="%ATTACHURLPATH%/BLAST.JPG" alt="BLAST.JPG" width="745" height="497" />

---++ 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).

Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
jpg	BLAST.JPG	r1	manage	217.2 K	2020-12-03 - 21:15	ElizabethRobinson	BLAST result for iPseudomonas syringae/i
jpg	Ribosomal_Database_Project.JPG	r1	manage	135.9 K	2020-12-03 - 21:10	ElizabethRobinson	SeqMatch results for Pseudomonas syringae

This topic: Lab > WebHome > ProtocolList > Protocols16SSequencing
Topic revision: r8 - 2020-12-03 - ElizabethRobinson