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Extracting phage genomic DNA

Objective

To get genomic DNA from phage

Rationale

Once you have a phage lysate, it is useful to extract phage DNA so that it can be sequenced later on. In this protocol we’re using a kit formulated to extract nucleic acids from viral samples. The process goes like this: The viral particles are first opened using Proteinase K, a Lysis Buffer and heat. Then, ethanol is added and the sample is loaded into a column with a silica matrix. The DNA molecules bind to the silica matrix, and the impurities such as proteins and nucleases are removed by washing them off with the Wash Buffer. Finally the DNA is recovered from the column with molecular grade water, which then we can store and use for sequencing later on.


Materials

  • 200 μL of cell-free phage lysate
  • 250 μL of 96-100% molecular grade ethanol
  • 20 μL of molecular grade water
  • 2 microcentrifuge tubes
  • PureLink™ Viral RNA/DNA Mini Kit
    • 200 μL of Lysis Buffer
    • 25 μL of Proteinase K
  • 1000 μL of Wash Buffer WII
    • 1 Viral Spin Column
    • 3 Collection tubes
  • Heat block
  • Vortex
  • Microcentrifuge

Before you start:

  • Make sure you have at least 200 μL of your phage lysate, at a concentration of at least 108 pfu/mL. You need a high concentration stock for this protocol to work.
  • Labeling your tubes is particularly important! We recommend you label 2 microcentrifuge tubes, and 1 viral spin column before starting.

Procedure

  1. 🌡️ Set the heating block to 56 ℃.
  2. ➕ Add 200 μL of your Phage Lysate into a clean and labeled microcentrifuge tube.
  3. ➕ Add 25 μL of Proteinase K to the same microcentrifuge tube.
  4. ➕ Add 200 μL of Lysis Buffer to the same microcentrifuge tube.
  5. 🌪 Mix the contents of the microcentrifuge tube by vortexing for 15 seconds.
  6. ⏳ Incubate the tube in the heat block, at 56 ℃ for 15 minutes.
  7. ➕ Add 250 μL of 96-100% molecular grade ethanol.
  8. 🌪️ Mix the contents of the microcentrifuge tube by vortexing for 15 seconds.
  9. ⏳ Incubate the tube at room temperature for 5 minutes.
  10. Place a labeled viral spin column on a collection tube.
  11. Pipette all the contents of the microcentrifuge tube into the viral spin column.
  12. 💫 Centrifuge the viral spin column in the collection tube at 6800 x g for 1 minute.

    Your sample is now in the viral spin column

  13. Place the viral spin column in a new collection tube, you can discard the old collection tube (which now contains the liquid that went through the column)
  14. ➕ Add 500 μL of Wash Buffer WII into the viral spin column.
  15. 💫 Centrifuge the viral spin column in the collection tube at 6800 x g for 1 minute.
  16. Discard the contents of the collection tube (the liquid that went through the column)
  17. Repeat Steps 14-16
  18. Place the viral spin column in a new and clean collection tube. You can discard the old collection tube.
  19. 💫 Centrifuge the viral spin column in the collection tube at maximum speed for 1 minute.

    This step might seem like you’re spinning empty tubes, but remember your sample is in the viral spin column. The idea here is to remove any residual ethanol. This step is very important, because ethanol contamination can be very problematic later on!

  20. Place the viral spin column in a new, clean, and labeled microcentrifuge tube.
  21. ➕ Add 20 μL of molecular grade water to the viral spin column.
  22. ⏳ Incubate at room temperature for 1 minute.
  23. 💫 Centrifuge the viral spin column in the microcentrifuge tube at maximum speed for 1 minute.

    Your sample is now in the microcentrifuge tube The water will elute the nucleic acid from the column, so your microcentrifuge tube now contains purified viral nucleic acids. You can discard the viral spin column now.

  24. Store your purified viral DNA in the 4 ℃ fridge for later use.