Microbiome DNA isolation

Obtaining DNA from swab sample

This is a protocol documents how to extract DNA from a swab collected using the Norgen swab collection and DNA preservation kit.

The full formal protocol is available on the Norgen website: Microbiome DNA Isolation kit.

Materials

  • Preserved DNA sample from swab
  • Lysis Additive A
  • Binding buffer I
  • 70% Ethanol
  • Binding Buffer B
  • Wash Solution A
  • Elution Buffer B
  • DNA spin columns
  • 65˚C water bath
  • Ice
  • 1.5ml tubes
  • Centrifuge

Steps

Protocol C

Part I - Preparing swab sample

  1. Add 100 μL of Lysis Additive A to the swab collection tube and vortex briefly.

  2. Incubate the swab collection tube at 65˚C for 5 minutes.

  3. Carefully remove the swab from the collection tube.

  4. Label a microcentrifugue tube and transfer up to 1 mL of the preserved sample to the microcentrifuge tube.

  5. Centrifuge the tube for 2 minutes at 20,000 × g (~14,000 RPM). A thin white layer will form on the top of the supernatant.

  6. Label a new microcentrifuge tube and carefully transfer 700 μL of supernatant, without the white layer debris, to the microcentrifuge tube.

  7. Add 100 μL of Binding Buffer I, mix by inverting the tube a few times, and incubate for 10 minutes on ice.

  8. Spin the lysate for 2 minutes at 20,000 x g (~14,000 RPM) to pellet any cell debris.

  9. Label a new microcentrifuge tube. Using a pipette, transfer up to 700 μL of supernatant (avoid contacting the pellet with the pipette tip) into the new microcentrifuge tube.

  10. Add an equal volume of 70% ethanol to the lysate collected above (100 μL of ethanol is added to every 100 μL of lysate). Vortex to mix.

Part II - binding DNA to column

  1. Assemble a spin column with one of the provided collection tubes.

  2. Apply 700 μL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM). Discard the flowthrough and reassemble the spin column with the collection tube.

    Note

    Ensure the entire lysate volume has passed through into the collection tube by inspecting the column. If the entire lysate volume has not passed, spin for an additional minute at 20,000 x g (~14,000 RPM).

  3. Repeat step 2 with the remaining volume of lysate mixture.

Part III - wash column

  1. Apply 500 μL of Binding Buffer B to the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM).

    Note

    Ensure the entire Binding Buffer B has passed through into the collection tube by inspecting the column. If the entire wash volume has not passed, spin for an additional minute.

  2. Discard the flowthrough and reassemble the spin column with its collection tube.

  3. Apply 500 μL of Wash Solution A to the column and centrifuge for 1 minute at 10,000 x g (~10,000 RPM).

  4. Discard the flowthrough and reassemble the spin column with its collection tube.

  5. Repeat steps 3 and 4.

  6. Spin the column for 2 minutes at 20,000 x g (~14,000 RPM) in order to thoroughly dry the resin. Discard the collection tube.

Part IV - Elute DNA

  1. Place the column into a fresh 1.7 mL Elution tube provided with the kit.

  2. Add 50 μL of Elution Buffer B to the column.

  3. Centrifuge for 1 minute at 425 x g (~2,000 RPM), followed by a 1 minute spin at 20,000 x g (~14,000 RPM). If the entire volume has not been eluted, spin the column at 20,000 x g (~14,000 RPM) for 1 additional minute.

  4. (Optional): An additional elution may be performed if desired by repeating steps 2 and 3 using 50 μL of Elution Buffer. The total yield can be improved by an additional 20-30% when this second elution is performed.

The purified genomic DNA can be stored at 2-8°C for a few days. For longer term storage, -20°C is recommended.