eDNA Filter Extraction

(using Qiagen blood and tissue kit)

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Filter Lysing

Need:

• sterile sissors and forcepts
• 50 ml tube with 10% bleach OR
• ethanol lamp with 100% ethanol + 50 ml tube with 70% ethanol & 50 ml tube of MilliQ water
• DI water spray bottle
• 2 ml tubes (with tapered not flat bottoms)
• ice container
• Qiagen blood and tissue kit (box labeled for eDNA use)
  • Buffer ATL (if there are precipitates heat at 56 C and mix gently)
  • Proteinase K

Notes:

• extract milliQ water filters with same steps to use as extraction negative control (check with Qubit afterwards)
  • usually two per experiment or field collection
    
• it is best to label tubes before starting lysis and extraction
  • lysis tubes: sample name
  • spin-column tubes: sample name
  • final storage tubes: samples name (eg. 9TW_2410_01 or UMMS_2409_01) usually on top & side; & DNA extraction date

Steps:

*BOLDED STEPS = modifications to Qiagen blood and tissue kit protocol

• 1. preheat thermomixer to 56 °C
• 2. clean area with 10% bleach, water, then 70% ethanol
• 3. prep ice container
• 4. label 2 ml tubes
• 5. add 360 µl of Buffer ATL & 40 µl of Proteinase K into labeled 2 ml tubes
• 6. clean instruments by soaking in 10% bleach, spraying with DI water (holding over waste container/glassware), then wiping down with kimwipe. (could also sterilize with DI water, 70% ethanol, then then running instrument through an ethanol lamp filled with 100% ethanol)
• 7. carefully remove filter from storage tube with forcepts and unroll until it is just folded in half
• 8. cut filters into 3 strips

• 9. place filter strips into 2ml tube with the Buffer ATL & Proteinase K
• 10. seal tube
• 11. vortex for 15 sec
• 12. store on ice until all filters have been cut up and transfered
• 13. repeat steps 6 through 12 for every filter (changing gloves between each filter)
• 14. Place samples on thermo-mixer set at 56 °C at 600 rpm (check after 2 mins to make sure everything is sealed)
• 15. incubate for at least 12–14 hr

DNA extraction

Need:

• 100% molecular grade ethanol
• Qiagen blood and tissue kit (box labeled for eDNA use)
  • DNeasy spin columns
  • DNeasy collection tubes
  • Buffer AL
  • Buffer AW1
  • Buffer AW2
• 1.5 or 2 ml microcentrifuge tubes (for final storage)
• warmed 1X low TE buffer (37ºC); may need to make by diluting 20X low TE buffer

Steps:

*BOLDED STEPS = modifications to Qiagen blood and tissue kit protocol

• 1. prep and clean bench with 10% bleach, water, then 70% ethanol
• 2. label spin-column tubes
• 3. remove samples from thermo-mixer
• 4. add 400 μl Buffer AL to samples, then vortex
• 5. add 400 μl Ethanol (100% molecular grade), then vortex
• 6. pipet half of solution to DNeasy Mini spin column placed in a 2 ml collection tube
• 7. centrifuge for 1 min at ≥ 6000 x g (8000 rpm)
• 8. change gloves after each centrifuge step
• 9. discard the flow-through & collection tube
• 10. tranfer spin column to a new collection tube
• 11. pipet the rest of the solution from the initial tubes into the spin column
• 12. centrifuge for 1 min at ≥ 6000 x g (8000 rpm)
• 13. discard the flow-through & collection tube
• 14. tranfer spin column to a new collection tube
• 15. add 500 μl Buffer AW1
• 16. centrifuge for 1 min at ≥6000 x g (8000 rpm)
• 17. discard the flow-through & collection tube
• 18. tranfer spin column to a new collection tube
• 19. add 500 μl Buffer AW2
• 20. centrifuge for 3 min at 20,000 x g (14,000 rpm)
• 21. discard flow through & spin again for 1 more min
• 22. make sure spin column membrane are dry after this step; spin again for 1 min if not
• 23. discard the flow-through & collection tube
• 24. tranfer spin column to a new 1.5 ml or 2 ml microcentrifuge tube
• 25. add 50 μl of warm low TE buffer (warm in thermomixer, 37 C)
• 26. incubate for 1 min at room temperature (15–25°C)
• 27. centrifuge for 1 min at ≥6000 x g (8000 rpm)
• 28. add another 50 μl warm low TE buffer
• 29. incubate for 10 min at room temperature (15–25°C)
• 30. centrifuge for 1 min at ≥6000 x g (8000 rpm)
• 31. store in fridge for a few days or freeze at -20 or -80 if not using immediately

Possible Next Steps

• Qubit
• need to dilute DNA? -> dilute with low TE buffer
• PCR then gel or qPCR
  • if doing PCR then gel, follow the qPCR profile and amounts (replace the probe with water, and use PCR strip tubes instead of a plate