Peatland Soil: NucleoMag vs PowerSoil Extraction Comparison

Owned by Shannon Harris
Last updated: Sept 29, 2021
3 min read

We will be extracting 47 soil samples (in duplicate) and one blank for each extraction method to determine the success of the NuceloMag extraction protocol.

MN NuceloMag DNA Microbiome Extraction Protocol

  1. Transfer up to 200mg of sample material to MN Bead Tube Type A. (done previously)

  2. Add 700uL of Lysis Buffer MI1.

  3. Add 150uL of Enhancer SX.

  4. Add 2.5uL of Liquid RNase A. (in question)

  5. Agitate on the mixer mill for 2min at 30Hz.

  6. Incubate the tubes for 5min at RT to allow fine debris to settle.

  7. Centrifuge for 5min at 11,000 x g. (If excessive foam formation occurs, centrifuge for an additional 5min)

  8. Transfer the supernatant to a new 2mL safe lock microcentrifuge tube.

  9. Add 150uL of Buffer MIc, close lid and vortex for 5s. (precipitation of contaminants)

  10. Incubate for 10min at 2-8°C. (preferably on ice but can also use refrigerator)

  11. Centrifuge for 5min at 11,000 x g.

  12. Transfer up to 500uL of lysate (supernatant) to Square-well block.

  13. NOTE: Resuspend beads before usage, homogenize bottle via vortex. Add 25uL of NucleoMag B-Beads and 310uL of Binding Buffer MI2. Pipette up and down 10x and incubate at RT for 5min. (Pipette gently to avoid foaming, if using electronic pipette, use only 40% of the total volume as mixing volume).

  14. Separate beads by placing the Square-well Block on the NucleoMag SEP magnetic separator. Wait 5min until all beads have been attracted to the magnets. Remove and discard supernatant by pipetting.

  15. Remove Square-well Block from the NucleoMag SEP magnetic separator.

  16. Add 600uL of Buffer MI3 to each well and resuspend the beads by either shaking or pipetting up and down until beads are resuspended completely (1-3min).

  17. Place Square-well Block back on the magnetic separator. Wait at least 2-5min. Remove and discard supernatant by pipetting.

  18. Repeat steps 15-17 once then move to step 19.

  19. Remove Square-well Block from the NucleoMag SEP magnetic separator.

  20. Add 600uL of Buffer MI4 to each well and resuspend the beads by either shaking or pipetting up and down until beads are resuspended completely (1-3min).

  21. Place Square-well Block back on the magnetic separator. Wait at least 2min then remove and discard supernatant by pipetting.

  22. Remove Square-well Block from the NucleoMag SEP magnetic separator.

  23. Add 600uL of 70% ethanol to each well and resuspend the beads by either shaking or pipetting up and down until beads are resuspended completely (1-3min).

  24. Place Square-well Block back on the magnetic separator. Wait at least 2min then remove and discard supernatant by pipetting.

  25. Air dry the magnetic beads for 10-15min at RT.

  26. Remove Square-well Block from the NucleoMag SEP magnetic separator.

  27. Add 100uL of Elution buffer MI5 to each well of the Square-well Block and resuspend the beads by shaking for 5min or repeated up and down pipetting. Incubate for 5-10min at RT.

  28. Place Square-well Block back on the magnetic separator. Wait at least 2min then transfer the supernatant containing the purified DNA to a suitable elution plate.

Qiagen PowerSoil Extraction Protocol

  1. Add up to 250mg of soil and 800uL of CD1. Vortex briefly to mix.

  2. Place tubes into mixer mill adapter and shake for 5min at 25Hz. Reorient the adapter so that the side closest to the machine body is now furthest from it. Shake again for 5min at 25Hz.

  3. Centrifuge at 15,000 x g for 1min.

  4. Transfer supernatant (approx. 500-600uL) to a clean 2mL microcentrifuge tube.

  5. Add 200uL of CD2 and vortex for 5sec.

  6. Centrifuge at 15,000 x g for 1min.

  7. Avoiding the pellet, transfer up to 700uL of the supernatant to a clean 2mL microcentrifuge tube. (Expect supernatant to be around 500-600uL).

  8. Add 600uL of CD3 and vortex for 5sec.

  9. Load 650uL of the lysate onto an MB Spin Column and centrifuge at 15,000 x g for 1min.

  10. Discard flow-through and repeat step 8 until all lysate has passed through the spin column.

  11. Carefully place the MB Spin Column into a clean 2mL collection tube.

  12. Add 500uL of solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1min.

  13. Discard flow-through and place the MB Spin Column back into the same 2mL collection tube.

  14. Add 500uL of C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1min.

  15. Discard flow-through and place the MB Spin Column into a new 2mL collection tube.

  16. Centrifuge at 16,000 x g for 2min.

  17. Carefully place the MB Spin Column into its corresponding labelled 1.5mL elution tube.

  18. Add 100uL of C6 to the center of the white filter membrane.

  19. Centrifuge at 15,000 x g for 1min. Discard the MB Spin Column after DNA is eluted.

Results

Comparison of each extraction method will be checked via DNA quantification on the synergy HTX. Spot checking of 260/230 ratios will be completed using the nanodrop.