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GenElute™ Bacterial Genomic DNA Kit (NA2100, NA2110, NA2120), Experienced User Protocol

For experienced users, an abbreviated protocol is listed below. For data, images, troubleshooting and other information.

Gram-Negative Bacterial Preparation

  1. Harvest Cells
    • Pellet 1.5 mL of bacterial broth culture at 12,000–16,000 × g for 2 minutes, discard media. When using enriched media, please see Procedure, step 1a.
  2. Resuspend Cells
    • Resuspend pellet in 180 μL Lysis Solution T.
      Optional: Add 20 μL RNase A, incubate RT for 2 minutes.
  3. Lyse Cells
    • Add 20 μL Proteinase K to cell suspension, vortex or pipet to mix. Incubate at 55 °C for 30 min.
    • Add 200 μL Lysis Solution C, vortex or pipette to mix. Incubate at 55 ºC for 10 minutes.
  4. Prepare Column
    • Add 500 μL of Column Preparation Solution to each binding column.
    • Spin at ≥12,000 × g for 1 minute. Discard flow-through.
  5. Bind DNA to Column
    • Add 200 μL ethanol to the lysed cells, vortex or invert to mix.
    • Transfer EtOH mixture to binding column. Spin at ≥ 6500 × g for 1 minute.
  6. Wash Column
    • Transfer column to new collection tube. Add 500 μL Wash Solution 1 to column. Spin at ≥ 6500 × g for 1 minute.
    • Transfer column to new collection tube. Add 500 μL Wash Solution Concentrate to column. Spin at ≥12,000 × g for 3 minutes to dry column.
  7. Elute DNA
    • Transfer column to new collection tube. Add 200 μL of Elution Solution. Spin at ≥ 6500 × g for 1 minute.
      Optional: Repeat in new or same tube.

Gram-Positive Bacterial Preparation

  • Prepare Lysozyme Solution (Lysozyme sold separately — Catalog No. L4919)
    Prepare a 2.115 × 106 unit/mL Lysozyme Solution using the included Gram-Positive Lysis Buffer as the diluent. 200 μL of Lysozyme Solution is needed for each prep. Make extra to account for pipetting error.
  1. Harvest Cells
    • Pellet 1.5 mL of bacterial broth culture at 12,000–16,000 × g for 2 minutes, discard media. When using enriched media, please see Procedure, step 2b.
  2. Digest Cell Wall
    • Resuspend pellet in 200 μL Lysozyme Solution and incubate at 37 °C for 30 minutes.
      Optional: Add 20 μL RNase A, incubate RT for 2 min.
  3. Lyse Cells
    • Add 20 μL Proteinase K and 200 μL Lysis Solution C to cell suspension, vortex or pipette to mix. Incubate at 55 °C for 10 minutes.
  4. Prepare Column
    • Add 500 μL of Column Preparation Solution to each binding column.
    • Spin at ≥12,000 × g for 1 minute. Discard flow-through.
  5. Bind DNA to Column
    • Add 200 μL ethanol to the lysed cells, vortex or invert to mix.
    • Transfer EtOH mixture to binding column. Spin at ≥ 6500 × g for 1 minute.
  6. Wash Column
    • Transfer column to new collection tube. Add 500 μL Wash Solution 1 to column. Spin at ≥ 6500 × g for 1 minute.
    • Transfer column to new collection tube. Add 500 μL Wash Solution Concentrate to column. Spin at ≥12,000 × g for 3 minutes to dry column.
  7. Elute DNA
    • Transfer column to new collection tube. Add 200 μL of Elution Solution. Spin at ≥ 6500 × g for 1 minute.
      Optional: Repeat in new or same tube.
gDNA-graph

Storage and Stability

Store the kit at room temperature. If any kit reagent forms a precipitate, warm at 55–65 °C until the precipitate dissolves and allow to cool to room temperature before use.

Precautions and Disclaimer

The GenElute Bacterial Genomic DNA Kit is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet (SDS) for information regarding hazards and safe handling practices.

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