Category: Principles of Living Systems

DNA Extraction Protocol

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For POLS Lab

There’s more than one way to isolate DNA from bacteriophages.  The SEA-PHAGES program recommends using a kit for doing the isolation.  The video shows the DNA extraction method that uses the kit.  However, at Montana Tech, students have been using a different protocol for many years.

The quiz will rely heavily on the reading assignment from the phage discovery guide (pages 72-76).

Protocol 6.3: Collecting Phage Lysate

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For POLS Lab

Once you have taken your bacteriophage sample through two rounds of purification, it is time to amplify your clonal virus population.  This process involves first determining the titer of your purified phage.  (We will use a spot test of serially-diluted samples for this.)  Then, using the titer information, we will perform full-plate plaque assays using diluted viruses that should create a “webbed plate”.

Once the plaques on the webbed plates have grown to an appropriate size, that is that they have killed most of the Mycobacterium smegmatis in the top agar, it is time to collect the bacteriophage.  This is the best time to collect the bacteriophage because the recoverable virus titer should be highest at this point.

The video clearly shows the procedure of collecting the plate lysate.  You will see that once the lysate is drawn up into the syringe, it is then sent through a sterile filter.  As you know, this process will allow virus to pass through the filter but not any bacteria.

The purified and amplified bacteriophage will need to be titered at this point.  Our goal is to collect lysate that has greater than 5 x 10^9 pfu/ml (plaque-forming units per milliliter).  This “high titer” bacteriophage is necessary for the characterization steps of this process.

POLS Lab Poster Advice

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From the syllabus:

Scientific information is often shared and discussed at scientific meetings.  The final assignment for this class will be to present your research at Techxpo on Thursday, April 26, 2018.  While you will make and present one poster with your partner, the four 15-point assignments (use Microsoft Word) will be created individually and graded separately.  The merging and formatting of your ideas with your partner’s ideas (use Microsoft PowerPoint set to poster size) will be graded as one assignment.

  1. Background: (~200-300 words)
    • Three paragraphs with 3-5 sentences packed with information (just the facts)
    • Paragraph topic: Bacteriophage, host bacteria (ex. Mycobacterium smegmatis)
  2. Isolation: (~100 words)
    • One paragraph describing your isolation method and purification steps
    • Flowchart of steps (use your paragraph to explain the flowchart)
  3. Amplification: Not applicable to everyone (~100 words)
    • One paragraph describing your titer information to describe the steps following purification through the creating a webbed plate and determining the titer from your webbed plate
    • Include plaque assay images with a summary of calculations
  4. Characterization:
    • Unfortunately, not applicable
  5. Abstract: (~150-200 words)
    • One paragraph that condenses/summarizes the most important points from background, isolation, amplification (if applicable), and future directions sections.
  6. Future Directions (~200-300 words)
    • 2-3 paragraphs describing future directions flow chart
    • Future directions flow chart
  7. Title and names: 
    • A descriptive title that includes the name of your bacteriophage isolate
    • Group members in alphabetical order, then TA name, then Abby/Joel
    • Finally, add “Montana Tech of the University of Montana”
  8. Format:
    • Poster size = 3 feet by 4 feet
    • Background color: white
    • Readable (and consistent) font sizes