meth_3.1.3 Qiagen DNeasy PowerSoil modified for marine sediments Marine Microbes Coastal Project

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AM Project ID: Legacy

Extract DNA from frozen sediment samples (1.5 g) with Qiagen DNeasy® PowerSoil® Kit following manufacturer’s recommendation for extraction protocol except with additional purification steps:

  1. In PCR plate, pipette from aliquots, 100 μL of AMPure solution. Gently shake before to resuspend beads.
  2. Add ~100 μL sample and pipette to mix. Mix gently as fast pipetting can displace sample from well.
  3. Leave to stand for a minimum of 10 min to allow strands to bind to magnet beads.
  4. Transfer PCR plate to SuperMagnet. Leave for 10 min for rings to form.
  5. Carefully remove solution in wells without disturbing the ring of beads.
  6. Add 200 μL 70% ethanol and leave for 30 s.
  7. Careful not to disturb the bead ring, pipette mix the ethanol to wash and remove from sample. Repeat for a total of 3 washes.
  8. After the last wash, make sure all ethanol is pipetted out from wells.
  9. Allow to air dry for 9-10 min. Do NOT allow beads to dry!
  10. Remove plate from magnet. Add 100 μL nuclease free water for DNA and pipette to elute beads and strands. Elution is rapid.
  11. Place plate back on magnet for 10 min.
  12. Carefully pipette out the sample into 1.5 mL sterile tubes labelled earlier and place on ice.
  13. If you suspect carryover of magnet beads, place tube back on magnet plate and being aliquoting 50 μL DNA to remaining 1.5 mL tube. With care, a final drop of sample containing magnet beads can be separated and removed.
  14. Place 1 x 50 μL DNA aliquot at -20 °C. Keep 1 x 50 μL DNA aliquot “dirty” sample on ice to nanodrop.
  15. The final DNA should be labelled according to the instruction given in the spreadsheet “sampling_schedule” and stored at -80 °C.