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

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

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

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

CREATE 10N NaOH; final volume: 40mL

MIX Inhibitor Removal Wash Buffer; final volume: 500mL

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

WEIGH sediment samples into a centrifuge-safe tube

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

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

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

DISCARD supernatant

REPEAT steps 6 and 7 three to four times

PROCEED with DNA extractions

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

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

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

TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube

ADD 200µL of Solution CD2

VORTEX briefly to mix

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

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

ADD 600µL of Solution CD3

VORTEX briefly to mix

LOAD 650µL of the lysate onto a MB Spin Column

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

DISCARD the liquid flow-through

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

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

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

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

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

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

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