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gDNA_Isolation_Mechanical.md

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Genomic DNA Purification From Gram-Positive Bacteria

Theory

This protocol has been adapted from Qiagen's DNeasy UltraClean Microbial Kit protocol. The goal of using this kit is to extract higher concentrations of gDNA from gram-positive bacteria for the purpose of whole genome sequencing using mechanical lysis. This protocol is generally prefered relative to the enzymatic protocol in this repository.

Materials

  • DNeasy UltraClean Microbial Kit (12224-250) Note: there is also a 96 well plate versio nof this kit
  • Nuclease-free water
  • Centrifuge
  • Pipettes
  • Vortex
  • Bead Beater (Ex. MPBio fastprep-96)
  • Thermal mixer or heating block

Protocol

Notes before starting:

  • If Solution SL has precipitated, heat at 55˚C for 5-10 minutes
  • Shake to mix Solution SB before use

Protocol:

  1. Add 1.8 mL of microbial culture to a 2 mL collection tube and centrifuge at 10,000 x g for 30 seconds at room temperature. Decant the supernatant and spin the tubes again at 10,000 x g for 30 seconds at room temperature. Completely remove the supernatant with a pipette tip.
  2. Resuspend the cell pellet in 300 µl of PowerBead Solution and gently vortex to mix. Transfer resuspended cells to PowerBead Tube.
  3. Add 50 µl of Solution SL to the PowerBead Tube.
  4. Incubate at 70˚C for 10 minutes/
  5. Place the PowerBead Tubes in the bead beater. Beat for one minute at max speed, followed by a one minute break. Complete this for a total of five minutes.
  6. Place the PowerBead Tubes in a centrifuge and centrifuge at a maximum of 10,000 x g for 30 seconds at room temperature.
  7. Transfer the supernatant to a clean 2 mL collection tube.
  8. Add 100 µl of solution IRS to the supernatant and vortex for 5 seconds. Incubate at 4˚C for 5 minutes.
  9. Centrifuge the tubes at 10,000 x g for 1 minute at room temperature.
  10. Avoiding the pellet, transfer the entire volume of supernatant to a clean 2 mL collection tube.
  11. Add 900 µl of Solution SB to the supernatant and vortex for 5 seconds.
  12. Load about 700 µl into a MB Spin Column and centrifuge at 10,000 x g for 30 seconds at room temperature. Discard the flow-through, add the remaining supernatant to the MB Spin Column, and centrifuge again at 10,000 x g for 30 seconds at room temperature. *Note: Each sample processed will require 2-3 loads. Discard all flow-through liquid. A vacuum manifold may also be used here.
  13. Add 300 µl of Solution CB and centrifuge at 10,000 x g for 1 minute at rooom temperature.
  14. Discard the flow-through. Centrifuge at 10,000 x g for 1 minute at room temperature.
  15. Place the MB Spin Column in a new 2 mL collection tube.
  16. Add 50 µl of nuclease-free water to the center of the white filter membrane.
  17. Centrifuge at 10,000 x g for 30 seconds at room temperature.
  18. Discard the MB Spin Column. The DNA is now ready for downstream applications. Store between -20 to -80˚C.
  19. Quality control by measuring concentration on Nanodrop.