Identification of unknown bacterial isolates using Sanger sequencing of the 16S rRNA gene
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Before starting

Materials

  • Promega GoTaq Green Master Mix (-20C)
  • Molecular biology grade, nuclease-free water (-20C or room temperature)
  • 27F Primer
  • 1492R primer
  • 515F primer (optional)
  • QIAGEN QIAquick PCR purification kit
  • 100% Ethanol

Step 1: Obtain genomic DNA

  • Extract DNA using QIAGEN DNeasy kit (bacterial protocol) or other method of bacterial DNA extraction.
  • Quantify DNA using the Qubit.

Step 2: PCR Amplify the full-length 16S rRNA gene

  • Prepare the following reaction mixture in each PCR well/tube

  • Place in thermocycler and run the following program under CHMI -> PCR -> 16SFULL (bold indicates 30 cycles)

Component Volume Final Concentration
GoTaq Green Master Mix, 2X 25 uL 1X
27F primer, 10 uM 1 uL 0.2 uM
1492R primer, 10 uM 1 uL 0.2 uM
DNA template 1-5 uL ~150 ng template (< 250 ng)
Nuclease-free water to 50 uL NA
Temp (C) Time (min:sec)
95 2:00
95 0:30
55 0:30
72 1:40
72 5:00
12 hold

Step 3. Clean up the PCR products

  • Follow the QIAGEN QIAquick PCR purification protocol.

Step 4: QC on purified PCR products

  • Perform Qubit quantification on all samples

  • Optional: Select a few samples and run an agarose gel or tapestation (DNA 5000 tape) to ensure there is a major product at approximately 1400 bp.

Step 5: Submit PCR products for sequencing

  • Follow the submission guide instructions carefully for submitting to the UPenn DNA sequencing facility

  • Each tube should contain the following:

    • 6 uL of PCR product at 25 ng/uL
    • 3 uL of the desired primer at 1.1 uM
    • If your DNA is not sufficiently concentrated, just submit the sample anyway, your results may be fine.
    • The sequencing facility suggests 10 ng of DNA per 100 bp.

Step 6: Interpret results

  • Assemble the sequencing reads using your favorite assembly software (Geneious has an easy-to-use interface).

  • Obtain the full length, consensus sequence seach for the best match using either BLAST or a recent tool called Bitsliced Genomic Signature Index (BIGSI) which is available here and you can read more about on the Github page for BIGSI

Tags: microbiome