Home Protocols Protocol for Genomic DNA Extraction from Gram-positive Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch® Genomic DNA Purification Kit (NEB #T3010)

Protocol for Genomic DNA Extraction from Gram-positive Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch® Genomic DNA Purification Kit (NEB #T3010)

Overview

This protocol enables the genomic DNA extraction from up to 2 x 109 gram-poitive bacteria. This protocol can successfully extract genomic DNA from B.subtilis and B.thuringiensis, but needs to be tested for other gram-positive bacteria on case-by-case basis.

Materials needed:

  • 50 mM Tris-HCl, 0.5 mM EDTA pH 7.5 is required (not supplied). Alternatively, TE Buffer can be used.
  • NEBExpress T4 Lysozyme (NEB #P8115)

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.
  • Set a thermal mixer (e.g., ThermoMixer® or similar device), or a heating block to 56°C for sample lysis.
  • 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.
  • Do not load a single column with the lysed sample more than once; over-exposure of the matrix to the lysed sample can cause the membrane to expand and dislodge.

 

Genomic DNA Extraction Consists of Two Stages:

PART 1: Sample Lysis

PART 2: Genomic DNA binding and elution

 

Part 1: Sample Lysis

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

  2. Add 100 μl of 50 mM Tris-Cl, 0.5 mM EDTA pH 7.5 (or TE Buffer) and resuspend bacterial pellet by pipetting or vortexing.

  3. Add 10 μl T4 Lysozyme (NEB #P8115) and mix by pipetting or vortexing briefly. Incubate at room temperature for 5 minutes. Lysates may remain turbid to some degree.

  4. Add 10 μl Proteinase K and 100 μl Tissue Lysis Buffer and vortex briefly. Incubate at 56°C for a minimum of 30 minutes in a thermal mixer with agitation at full speed (1400 rpm or 2000 rpm if available). If lysates were not clear after T4 Lysozyme incubation, they will soon become clear after adding the Proteinase K and Tissue Lysis Buffer if T4 Lysozyme works for the respective bacteria species. If a thermal mixer is not available, vortex samples several times during incubation and continuously for 1 minute at the end of the incubation.

  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 or 2000 rpm if available).

  6. Proceed to Genomic DNA binding and elution.


Part 2: 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. Do not transfer any foam. Proceed immediately to step 3. Do not reload the same column with more sample; over-exposure of the matrix to the lysed sample can cause the membrane to expand and dislodge.

    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 2,000 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 60°C for 5 minutes. 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%). Incubation at 60°C for 5 minutes will give higher yields as it helps elute a larger proportion of the tough-to-elute long gDNA fragments.

    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. Discard spin column and close cap. Vortex eluate briefly for homogeneity.