[Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Optional Concentration
Grayson Huston
Abstract
Protocol (both increased sediment amount up to 2.0g as well as concentrating DNA post-extraction) unsuccessful at detecting fish sedDNA from lakes in Maine, USA.
Both protocols successful at detecting fish sedDNA collected in streams during anadromous fish sea-run migrations
Steps
Modified PowerSoil Pro extraction - sample preparation & cell lysis
CENTRIFUGE sediment samples briefly to separate pore water
DISCARD pore water to retain only sediment samples
SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom
ADD up to 2.0g of wet sediment to the PowerBead Pro Tube
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
Modified PowerSoil Pro extraction - inhibitor removal
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
Modified PowerSoil Pro extraction - bind DNA
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 8 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
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
Modified PowerSoil Pro extraction - elute the DNA
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
(Optional) DNA Concentration with PALL Nanosep 30K Centrifugal Devices
ENSURE that the sample reservoir is firmly placed into the filtrate receiver
PIPETTE 50-100µL of DNA extract into the sample reservoir
CAP the Nanosep device
CENTRIFUGE at 5000x g for 0h 2m 0s
RECOVER concentrated sample from the sample reservoir with a micropipette
TRANSFER concentrated sample to a new 1.5 mL Microcentrifuge Tube
Concentrated DNA is now ready for downstream applications
