[Modified] Lake ABPS Protocol - University of Maine

CENTRIFUGE sediment samples at 5250x g for 0h 5m 0s

DISCARD pore water using a sterile pipette, so only sediment remains

ADD 10g of wet sediment to a sterile 50 mL tube

ADD 6mL sodium hydroxide (NaOH, 0.33M) to the sample

ADD 3mL Tris-EDTA (pH 8.0) to the sample

VORTEX sample at max speed for 0h 1m 0s

INCUBATE sample at 65°C for 0h 55m 0s

ALLOW samples to cool to Room temperature

CENTRIFUGE samples at 5250x g for 1h 0m 0s

TRANSFER 7.5mL of supernatant to a new, sterile 50 mL tube

ADD 7.5mL Tris-HCl (pH 6.7)

ADD 1.5mL sodium acetate (3M, pH 5.2)

ADD 30mL molecular grade ethanol

INCUBATE samples at -20°C for 1h 0m 0s

CENTRIFUGE sample at 5250x g for 1h 0m 0s

DISCARD supernatant

ALLOW remaining ethanol to evaporate off of concentrated pellet before proceeding to next step

WEIGH the concentrated pellet and split it into multiple 0.5g replicates

TRANSFER each 0.5g replicate into a PowerBead Pro Tube

ADD 800µL of Solution CD1 to each PowerBead Pro Tube

SECURE PowerBead Pro Tubes horizontally to a Vortex Adapter

VORTEX for 0h 10m 0s

ROTATE tubes so caps are oriented in the opposite direction

VORTEX for another 0h 10m 0s

CENTRIFUGE sample at 15000x g for 0h 2m 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 lysate onto an MB spin column

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

DISCARD the liquid flow-through

REPEAT step 14 to ensure all the lysate has passed through the MB Spin Column

CAREFULLY place the MB spin column into a clean 2mL 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 the 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 2mL Collection Tube

ADD 50-100µL of Solution C6 to the center of the white membrane in the MB Spin Column

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

DISCARD the MB Spin Column

POOL all replicates into a sterile 1.5 mL Microcentrifuge Tube

DNA is now ready for downstream applications