Genomic DNA Purification from Gram-negative Bacteria (NEB #T3010)

Up to 2 x 109 Gram-negative bacteria can be processed using either a quick protocol which employs Lysozyme for bacterial cell wall lysis, or a longer protocol that does not require enzymatic lysis with Lysozyme. Both protocols are available below.

Before You Begin:

  • Store RNase A and Proteinase K at -20°C.
  • Add ethanol (≥ 95%) to the Monarch gDNA Wash Buffer concentrate as indicated on the bottle label.
  • Cold PBS or 10 mM Tris-HCl pH 8.0 is required (not supplied).
  • Set a thermal mixer (e.g. ThermoMixer or similar device), or a heating block to 56°C for sample lysis.
  • For Lysozyme-based Lysis:
    • Set a thermal mixer or heating block to 37°C.
    • Prepare or thaw a stock solution of Lysozyme (not supplied) (25 mg/ml in water or 10 mM Tris-Cl, pH 8.0)
  • To prepare for elution, set a heating block to 60°C. Preheat the appropriate volume of elution buffer to 60°C (35–100 μl per sample). Confirm the temperature, as temperatures are often lower than indicated on the device.

Rapid Protocol (requires Lysozyme)

  1. Harvest a maximum of up to 2 x 109 Gram-negative bacteria by centrifugation for 1 minute at > 12,000 x g. Discard supernatant.

  2. Add 90 μl of cold PBS or 10 mM Tris-Cl pH 8.0 and resuspend bacterial pellet by vortexing.

  3. Add 10 μl Lysozyme solution (25 mg/ml) and vortex briefly, then add 100 μl Tissue Lysis Buffer and vortex thoroughly.

  4. Incubate at 37°C for 5 minutes or until clear. Most lysates will become fully clear, but for some bacteria a slight haze may remain.

  5. Add 10 μl Proteinase K, vortex briefly, and incubate at 56°C for a minimum of 30 minutes in a thermal mixer with agitation at full speed (~1400 rpm).

  6. Add 3 μl of RNase A to the lysate, vortex briefly, and incubate for a minimum of 5 minutes at 56°C with agitation at full speed (~1400 rpm).

  7. Proceed to Genomic DNA Binding and Elution.

Simplified Protocol (no Lysozyme required)

  1. Harvest a maximum of up to 2 x 109 Gram-negative bacteria by centrifugation for 1 minute at > 12,000 x g. Remove supernatant.

  2. Add 100 μl of PBS or 10 mM Tris-Cl pH 8.0 and resuspend bacterial pellet by vortexing.

  3. Add 10 μl Proteinase K and vortex briefly, then add 100 μl Tissue Lysis Buffer and vortex thoroughly.

  4. Incubate at 56°C for 1–4 hours in a thermal mixer with agitation at full speed until the lysate is mostly clear and ceases to change in appearance (lysis is usually complete within 2 hours).

  5. Add 3 μl of RNase A to the lysate, vortex briefly, and incubate for a minimum of 5 minutes at 56°C with agitation at full speed (~1400 rpm).

  6. Proceed to Genomic DNA Binding and Elution.

 

GENOMIC DNA BINDING AND ELUTION

  1. Add 400 μl gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for 5-10 seconds. Thorough mixing is essential for optimal results.

  2. Transfer the lysate/binding buffer mix (~600 μl) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 3. Avoid touching the upper column area with lysate/binding mix and avoid transferring foam that may have formed during lysis. Any material that touches the upper area of the column, including any foam, may lead to salt contamination in the eluate.

  3. Close the cap and centrifuge: first for 3 minutes at 1,000 x g to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for 1 minute at maximum speed (> 12,000 x g) to clear the membrane. Discard the flow-through and the collection tube. For optimal results, ensure that the spin column is placed in the centrifuge in the same orientation at each spin step (for example, always with the hinge pointing to the outside of the centrifuge); ensuring the liquid follows the same path through the membrane for binding and elution can slightly improve yield and consistency.

  4. Transfer column to a new collection tube and add 500 μl gDNA Wash Buffer. Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for 1 minute at maximum speed (12,000 x g), and discard the flow through. The collection tube can be tapped on a paper towel to remove any residual buffer before reusing it in the next step. Inverting the spin column containing wash buffer prevents salt contamination in the eluate.

  5. Reinsert the column into the collection tube. Add 500 μl gDNA Wash Buffer and close the cap. Centrifuge immediately for 1 minute at maximum speed (>12,000 x g), then discard the collection tube and flow through.

  6. Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included). Add 35-100 μl preheated (60°C) gDNA Elution Buffer, close the cap and incubate at room temperature for 1 minute. Elution in 100 μl is recommended, but smaller volumes can be used and will result in more concentrated DNA but a reduced yield (20–25% reduction when using 35 μl). Eluting with preheated elution buffer will increase yields by ~20–40% and eliminates the need for a second elution. For applications in which a high DNA concentration is required, using a small elution volume and then eluting again with the eluate may increase yield (~10%). The elution buffer (10 mM Tris-Cl, pH 9.0, 0.1 mM EDTA) offers strong protection against enzymatic degradation and is optimal for long term storage of DNA. However, other low-salt buffers or nuclease-free water can be used if preferred. For more details on optimizing elution, please refer to “Considerations for Elution & Storage” in the product manual.

  7. Centrifuge for 1 minute at maximum speed (> 12,000 x g) to elute the gDNA. 

 

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