TITLE: [Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume &amp; Optional Concentration
AUTHORS: Grayson Huston

[DESCRIPTION]
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]
SECTION: Modified PowerSoil Pro extraction - sample preparation &amp; cell lysis
1. CENTRIFUGE sediment samples briefly to separate pore water

DISCARD pore water to retain only sediment samples
SECTION: Modified PowerSoil Pro extraction - sample preparation &amp; cell lysis
2. SPIN the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom

ADD up to 2.0 g of wet sediment to the PowerBead Pro Tube
 
ADD 800 µL Solution CD1

VORTEX briefly to mix
SECTION: Modified PowerSoil Pro extraction - sample preparation &amp; cell lysis
3. SECURE PowerBead Pro Tubes horizontally to a 1.5mL-2.0mL Vortex Adapter

VORTEX for 10 min

ROTATE tubes so caps are oriented in opposite direction

VORTEX for another 10 min 
SECTION: Modified PowerSoil Pro extraction - sample preparation &amp; cell lysis
4. CENTRIFUGE PowerBead Pro Tube at 15000 x g for 1 min 

TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube
SECTION: Modified PowerSoil Pro extraction - inhibitor removal
5. ADD 200 µL of Solution CD2

VORTEX briefly to mix
SECTION: Modified PowerSoil Pro extraction - inhibitor removal
6. CENTRIFUGE at 15000 x g for 1 min 

AVOIDING the pellet, transfer all supernatant to a clean 2 mL Microcentrifuge Tube 
SECTION: Modified PowerSoil Pro extraction - bind DNA
7. ADD 600 µL of Solution CD3

VORTEX briefly to mix
SECTION: Modified PowerSoil Pro extraction - bind DNA
8. LOAD 650 µL of the lysate onto a MB Spin Column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through
SECTION: Modified PowerSoil Pro extraction - bind DNA
9. 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
SECTION: Modified PowerSoil Pro extraction - wash spin column
10. ADD 500 µL of Solution EA to the MB Spin column

CENTRIFUGE at 15000 x g for 1 min 

DISCARD the liquid flow-through and place the MB Spin Column into same 2 mL Collection Tube
SECTION: Modified PowerSoil Pro extraction - wash spin column
11. ADD 500 µL of Solution C5 to the MB Spin Column

CENTRIFUGE at 15000 x g for 1 min

DISCARD the liquid flow-through and place the MB Spin Column into a new 2 mL Collection Tube
SECTION: Modified PowerSoil Pro extraction - wash spin column
12. CENTRIFUGE at 16000 x g for 2 min

CAREFULLY place the MB Spin Column into a new 1.5mL Elution Tube
SECTION: Modified PowerSoil Pro extraction - elute the DNA
13. ADD 100 µL of Solution C6 to the center of the white membrane in the MB Spin Column

INCUBATE at Room temperature for 1 min 

CENTRIFUGE at  15000 x g for 1 min 
SECTION: Modified PowerSoil Pro extraction - elute the DNA
14. 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 1 min

CENTRIFUGE at  15000 x g for 1 min 

DISCARD the MB Spin Column

DNA is now ready for downstream applications
SECTION: (Optional) DNA Concentration with PALL Nanosep 30K Centrifugal Devices
15. ENSURE that the sample reservoir is firmly placed into the filtrate receiver

PIPETTE 50-100 µL of DNA extract into the sample reservoir

CAP the Nanosep device
SECTION: (Optional) DNA Concentration with PALL Nanosep 30K Centrifugal Devices
16. CENTRIFUGE at 5000 x g for 2 min 

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