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Landscape of Interacting Microbes - DNA Extraction Protocols

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Extraction of DNA from environmental samples with ZymoBIOMICS™ DNA Miniprep Kit

Product info: https://www.zymoresearch.com/products/zymobiomics-dna-miniprep-kit

Protocol written by Shira Jaye Anteby-Maleh

Preparation

  1. Prepare and label the following tubes:
    • 1 set of bead tubes
    • 3 sets of collection tubes
    • 2 sets of 1.5ml microcentrifuge tubes
    • 1 set of Zymo-Spin IIIF (pink) columns
    • 1 set of Zymo-Spin IICR filters (white)
    • 1 set of Zymo-III HRC filters (white)
    • Total: 6 tubes and 3 filters per sample
  2. Create labels for final DNA tubes (side and top) using ToughTags
  3. Pipette 750ul (using P1000) lysis solution to each bead tube and 1.5ml (using 5ml pipette) RNA-later purified water into cryopreservation tubes (orange caps)

Bead beating

  1. Weigh out ~0.2500g sediment or ~0.2000g seagrass (leaf, root, and rhizome) and place in corresponding bead tube
  2. place remainder of sample in orange tubes for preservation at -80C)
  3. Centrifuge bead tubes for 18 min using program #1 (freeze after at -80C if extraction is on a different day):
    • Beat at 6 m/s for 1 min
    • Rest for 5 min
    • Repeat for 2 more cycles (3 times total)
  4. Microfuge at 10,000xg for 1 min (DNA is now in the supernatant)

DNA binding and washing

  1. Pipette 400ul (using P1000) supernatant into Zymo-Spin IIIF (pink) columns within collection tubes (try to pipette out as much supernatant as possible)
  2. Microfuge at 8,000xg for 1 min
  3. Discard Zymo-Spin IICR (pink) columns (DNA is in the flowthrough) (steps 6-8 are only repeated if there is a lot more than 400ul supernatant)
  4. Pipette 1,200ul (using 5ml pipette) DNA binding buffer to each collection tube with supernatant/DNA and gently mix up and down
  5. Pipette 800ul (using P1000) of mixture from step 9 into Zymo-Spin™ IICR Column (white) in new collection tubes
  6. Microfuge at 10,000xg for 1 min
  7. Discard flowthrough (DNA is in the filter)
  8. Repeat steps 10-12 with the remaining 800ul of the mixture from step 9 into the same Zymo-Spin™ IICR Column (white) within the same collection tubes
  9. Pipette 400ul (using P1000) wash buffer 1 into the same Zymo-Spin™ IICR Column (white)
  10. Microfuge at 10,000xg for 1 min
  11. Discard flowthrough
  12. Pipette 700ul (using P1000) wash buffer 2 into the same Zymo-Spin™ IICR Column (white)
  13. Microfuge at 10,000xg for 1 min
  14. Discard flowthrough
  15. Pipette 200ul (using P1000) wash buffer 2 into the same Zymo-Spin™ IICR Column (white)
  16. Microfuge at 10,000g for 1 min
  17. Discard flowthrough

DNA elution and purification

  1. Pipette 50ul (using P200) DNAse/RNAse-free water into the same Zymo-Spin™ IICR Column (white)s and place in new 1.5ml DNA microfuge tubes with caps
  2. Leave out for 1 min
  3. Microfuge at 10,000g for 1 min (eluted DNA is in the bottom of the 1.5ml tubes)
  4. Repeat the DNA elution (steps 23-25) using the flowthrough - pipette the flowthrough back onto the filter and spin
  5. Discard Zymo-II white filters
  6. Place Zymo-Spin III HRC (white) filters into new collection tubes and pipette 600ul HRC prep solution (using p1000) into filters
  7. Microfuge tubes from step 27 at 8,000xg for 3 min
  8. Discard HRC prep solution flowthrough
  9. Place Zymo-Spin III HRC (white) filter into new 1.5ml DNA microfuge tubes with caps (these tubes will have the final DNA)
  10. Pipette all 50ul (using P200) of the eluted DNA samples into the same Zymo-III white filters
  11. Microfuge tubes from steps 30-31 at 16,000g for 3 min
  12. Cap tubes, label, and freeze at -20C