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: First, we get rid of genetic material that is not from the phages with an enzyme treatment. Afterwards, the viral particles are opened using Proteinase K, a Lysis Buffer and heat. Then, an ethanol buffer 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 an elution buffer, which then we can store and use for sequencing later on.

Materials

  • 200 μL of cell-free phage lysate
  • 200 μL of 96-100% molecular grade ethanol
  • 2 microcentrifuge tubes
  • 5 μL of Digestion Enzyme Mix (DNase + RNase)
  • Qiagen™ DNeasy Kit
    • 180 μL of tissue Lysis Buffer (ATL)
    • 200 μL of Lysis Buffer (AL)
    • 20 μL of Proteinase K
    • 500 μL of ethanol Wash Buffer 1 (WI)
    • 500 μL of Wash Buffer 2 (WII)
    • 50 μL of elution buffer (AE)
    • 1 Viral Spin Column
    • 3 Collection tubes
  • Heat blocks
  • 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

Steps 1-6 will be done by instructors before class to cut down on incubation/waiting time.

  1. 🌡️ Set the heat blocks to 37 ℃ and 65 ℃.
  2. ➕ Add 200 μL of your Phage Lysate into a clean and labeled microcentrifuge tube.
  3. ➕ Add 5 μL of the Digestion Enzyme Mix into the same microcentrifuge tube.
  4. ⏳ Incubate tube at 37 ℃ for 30 minutes. During this step, the digestion enzymes will break down any bacterial DNA in the phage lysate.
  5. ⏳ Incubate tube at 65 ℃ for 20 minutes. This step deactivates the digestion enzymes, to ensure that they don’t degrade your phage DNA!
  6. 🌡️ Set one heat block to 80 ℃.

    Students start here with step 7!

  7. ➕ Add 20 μL of Proteinase K to your microcentrifuge tube.
  8. ➕ Add 180 μL of tissue Lysis Buffer (ATL) to your microcentrifuge tube.
  9. 🌪 Mix the contents of the microcentrifuge tube by vortexing for 15 seconds.
  10. ⏳ Incubate the tube in the heat block, at 80 ℃ for 15 minutes.
  11. ➕ Add 200 μL of Lysis Buffer (AL).
  12. ➕ Add 200 μL of 96-100% molecular grade ethanol.
  13. 🌪️ Mix the contents of the microcentrifuge tube by vortexing for 15 seconds.
  14. Label a viral spin column with a collection tube.
  15. Pipette all the contents of the microcentrifuge tube (approximately 800 μL) into the viral spin column
  16. 💫 Centrifuge the viral spin column in the collection tube at 6000 x g for 1 minute.

    Your sample DNA is now in the viral spin column

  17. 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).
  18. ➕ Add 500 μL of ethanol Wash Buffer 1 (WI) into the viral spin column.
  19. 💫 Centrifuge the viral spin column in the collection tube at 6000 x g for 1 minute.
  20. Place the viral spin column in a new and clean collection tube. You can discard the old collection tube.
  21. ➕ Add 500 μL of Wash Buffer 2 (WII) into the viral spin column.
  22. 💫 Centrifuge the viral spin column in the collection tube at maximum speed (20,000 x g) for 1 minute.
  23. 💫 Empty the collection tube in the biobin, then replace the column into the tube and spin at maximum speed (20,000 x g) for 1 minute to dry the column.
  24. Place the viral spin column in a new, clean, and labeled microcentrifuge tube.
  25. ➕ Add 50 μL of elution buffer (AE) to the viral spin column.

    Note: The elution buffer will be pre-warmed to 65 ℃ to increase yield.

  26. ⏳ Incubate at room temperature for 5 minutes.
  27. 💫 Centrifuge the viral spin column in the microcentrifuge tube at 6,000 x g for 1 minute.

    Your sample is now in the microcentrifuge tube The elution buffer 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.

  28. Measure the concentration on the NanoDrop machine.
  29. Store your purified viral DNA in the 4 ℃ fridge for later use.