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      [Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Inhibitor Removal Pre-Extraction

      Grayson Huston

      Published: 2023-04-06 DOI: 10.17504/protocols.io.yxmvm2be9g3p/v1
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      Abstract

      Protocol (wash buffer plus modified extraction) unsuccessful at detecting fish sedDNA from lakes in Maine, USA

      Protocol successful at detecting fish sedDNA collected from streams during an anadromous fish sea-run migration

      Steps

      Wash buffer reagents

      1.

      CREATE 0.5M EDTA, pH 8.0; final volume: 250mL

      Note
      ADD 52.025g Ethylenediamine Tetraacetic Acid, Tetrasodium Salt Dihydrate (EDTA) to a volumetric flaskADD 200mL DI water to dissolveTITRATE to pH 8.0 with Hydrochloric Acid (HCL, approx. 9.5 mL)ADD DI water to bring final volume to 250 mLAUTOCLAVE solution

      2.

      CREATE 1M Tris-HCl, pH 8.0; final volume: 500mL

      Note
      ADD 78.8g Tris-HCl to a volumetric flaskADD 400mL DI water to dissolveRAISE pH to 8.0 with 10N NaOHADD DI water to bring final volume to 500 mLAUTOCLAVE solution

      3.

      CREATE a batch of 0.5M Na2PO4*7H2O, pH 8.0; final volume: 250mL

      Note
      ADD 33.508g Na2PO4*7H2O to a volumetric flaskADD 200mL DI water and heat to dissolveRAISE pH to 8.0 with 10N NaOH (approx. 1.5 mL)ADD DI water to bring the final volume to 250 mLAUTOCLAVE solution

      4.

      CREATE 10N NaOH; final volume: 40mL

      Note
      ADD 16g NaOH to a volumetric flaskADD 40mL of DI water to dissolve

      5.

      MIX Inhibitor Removal Wash Buffer; final volume: 500mL

      Note
      10mL 0.5M EDTA, pH 8.025mL 1M Tris-HCl, pH 8.050mL Na2PO4*7H2P, pH 8.0415mL DI water

      AUTOCLAVE solution

      Final buffer contains 10mM EDTA pH 8.0 + 50mM Tris-HCl pH 8.0 + 50mM sodium phosphate dibasic heptahydrate (Na2PO4*7H2O) pH 8.0

      PCR inhibitor removal via wash buffer

      6.

      WEIGH sediment samples into a centrifuge-safe tube

      ADD approximately 3x the volume of Wash Buffer to the sediment samples

      Note
      Account for loss of sediment during wash process (e.g., wash 5g if you plan to extract 3g of sediment)If you measure 5g sediment, add ~15 mL of Wash Buffer

      7.

      VORTEX sample at maximum speed for 0h 0m 30s

      CENTRIFUGE at 1760x g for 0h 10m 0s

      DISCARD supernatant

      8.

      REPEAT steps 6 and 7 three to four times

      PROCEED with DNA extractions

      Modified PowerSoil pro extraction - sample preparation & cell lysis

      9.

      SPIN PowerBead Pro Tube briefly to ensure that all beads have settled at the bottom

      ADD 0.5g of washed sediment to the PowerBead Pro Tube

      ADD 800µL Solution CD1

      VORTEX briefly to mix

      10.

      SECURE PowerBead Pro Tubes horizontally to a 1.5mL-2.0mL Vortex Adapter

      VORTEX for 0h 10m 0s

      ROTATE tubes so caps are oriented in opposite direction

      VORTEX for another 0h 10m 0s

      11.

      CENTRIFUGE PowerBead Pro Tube at 15000x g for 0h 1m 0s

      TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube

      Modified PowerSoil pro extraction - inhibitor removal

      12.

      ADD 200µL of Solution CD2

      VORTEX briefly to mix

      13.

      CENTRIFUGE at 15000x g for 0h 1m 0s

      AVOIDING the pellet, transfer all supernatant to a clean 2 mL Microcentrifuge Tube

      Modified PowerSoil pro extraction - bind DNA

      14.

      ADD 600µL of Solution CD3

      VORTEX briefly to mix

      15.

      LOAD 650µL of the lysate onto a MB Spin Column

      CENTRIFUGE at 15000x g for 0h 1m 0s

      DISCARD the liquid flow-through

      16.

      REPEAT step 15 to ensure all of the lysate has passed through the MB Spin Column

      CAREFULLY place the MB Spin Column into a clean 2 mL Collection Tube

      Modified PowerSoil pro extraction - wash spin column

      17.

      ADD 500µL of Solution EA to the MB Spin column

      CENTRIFUGE at 15000x g for 0h 1m 0s

      DISCARD the liquid flow-through and place the MB Spin Column into same 2 mL Collection Tube

      18.

      ADD 500µL of Solution C5 to the MB Spin Column

      CENTRIFUGE at 15000x g for 0h 1m 0s

      DISCARD the liquid flow-through and place the MB Spin Column into a new 2 mL Collection Tube

      19.

      CENTRIFUGE at 16000x g for 0h 2m 0s

      CAREFULLY place the MB Spin Column into a new 1.5mL Elution Tube

      Modified PowerSoil pro extraction - elute the DNA

      20.

      ADD 100µL of Solution C6 to the center of the white membrane in the MB Spin Column

      INCUBATE at Room temperature for 0h 1m 0s

      CENTRIFUGE at 15000x g for 0h 1m 0s

      21.

      PIPETTE the liquid flow-through and re-add it to the center of the white membrane in the MB Spin Column

      INCUBATE at Room temperature for 0h 1m 0s

      CENTRIFUGE at 15000x g for 0h 1m 0s

      DISCARD the MB Spin Column

      DNA is now ready for downstream applications

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