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gDNA_Isolation_Enzymatic.md

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Genomic DNA Purification From Gram-Positive Bacteria

Theory

This protocol was adapted from New England BioLabs' Genomic DNA Purification from Gram-positive Bacteria and Archaea. The goal of using this kit is to extract higher concentrations of gDNA for the purpose of whole genome sequencing using enzymatic (as opposed to mechanical) lysis.

Materials

  • Monarch Genomic DNA Purification Kit (T3010L)
  • Lysozyme (25 mg/mL)
  • Mutanolysin (50 U/mL) Optional
  • PBS or 10 mM Tris-HCl pH 8.0
  • Thermal mixer or heating block
  • Microcentrifuge tubes
  • Nuclease-free water

Protocol

Part 1 - Lysis

  • Harvest 2 x 10 9 (~2-3 medium sized colonies or 1 mL liquid culture) gram-positive bacteria by centrifugation for 1 minute at >12,000 rpm. Remove supernantant.
  • Add 80 μl of PBS or 10 mM Tris-CI pH 8.0 and resuspend bacterial pellet by vortexing.
  • Add 20 μl Lysozyme solution and vortex briefly. Note: lysozyme may be supplemented with mutanolysin to improve extraction of lysozyme-resistant microbes like lactobacilli and some bifidobacteria.
  • Add 100 μl Tissue Lysis Buffer and vortex thoroughly.
  • Incubate at 37°C for 30 minutes.
  • Add 10 μl Proteinase K, vortex briefly
  • Incubate at 56°C for 30 minutes in a thermal mixer with agitation at full speed (~1400 rpm).
  • Add 3 μl of RNase A to the lysate, vortex briefly
  • Incubate for a minimum of 5 minutes at 56°C with agitation at full speed.
  • Proceed to Genomic DNA Binding and Elution.

Part 2- Clean Up

  • Add 400 μl gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for 5-10 seconds.
  • Transfer the lysate/binding buffer mix (~600 μl) to a gDNA Purification Column pre-inserted into a collection tube
  • Centrifuge for 3 minutes at 1,000 xg to bind the gDNA.
  • Centrifuge again for 1 minute at maximum speed (>12,000 xg) to clear the membrane.
  • Discard the flow-through and the collection tube.
  • Transfer column to a new collection tube and add 500 μl gDNA Wash Buffer. Close the cap and invert a few times, so the was buffer reaches the cap.
  • Centrifuge immediately for 1 minute at maximum speed (>12,000 xg), and discard the flow through.
  • Add 500 μl gDNA Wash Buffer and close the cap. Centrifuge immediately for 1 minute at maximum speed (>12,000 xg), then discard the collection tube and flow through.
  • Transfer the column into a DNase-free 1.5 mL microcentrifuge tube. Centrifuge for 1 minute at maximum speed (>12,000 xg) to pull off any additional ethanol.
  • Place the gDNA Purification Column into a new DNase-free 1.5 mL microcentrifuge tube.
  • Add 50 μl nuclease-free water to elute DNA. Close the cap and incubate at room temperature for 1 minute.
  • Centrifgue for 1 minute at maximum speed (>12,000 xg) to elute the gDNA.
  • Discard the filter column and label tube appropriately.
  • Store gDNA at -20°C for short-term storage.