TITLE: [Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume &amp; Inhibitor Removal Pre-Extraction
AUTHORS: Grayson Huston

[DESCRIPTION]
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]
SECTION: Wash buffer reagents
1. CREATE 0.5M EDTA, pH 8.0; final volume: 250mL
 

SECTION: Wash buffer reagents
2. CREATE 1M Tris-HCl, pH 8.0; final volume: 500mL
 

SECTION: Wash buffer reagents
3. CREATE a batch of 0.5M Na2PO4*7H2O, pH 8.0; final volume: 250mL

 


SECTION: Wash buffer reagents
4. CREATE 10N NaOH; final volume: 40mL
 

SECTION: Wash buffer reagents
5. 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
SECTION: 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
 

SECTION: PCR inhibitor removal via wash buffer
7. VORTEX sample at maximum speed for 30 s 

CENTRIFUGE at  1760 x g for 10 min 

DISCARD supernatant
SECTION: PCR inhibitor removal via wash buffer
8. REPEAT steps 6 and 7 three to four times

PROCEED with DNA extractions
SECTION: Modified PowerSoil pro extraction - sample preparation &amp; cell lysis
9. SPIN PowerBead Pro Tube briefly to ensure that all beads have settled at the bottom

ADD 0.5 g of washed 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
10. 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
11. 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
12. ADD 200 µL of Solution CD2

VORTEX briefly to mix
SECTION: Modified PowerSoil pro extraction - inhibitor removal
13. 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
14. ADD 600 µL of Solution CD3

VORTEX briefly to mix
SECTION: Modified PowerSoil pro extraction - bind DNA
15. 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
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
SECTION: Modified PowerSoil pro extraction - wash spin column
17. 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
18. 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
19. 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
20. 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
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 1 min

CENTRIFUGE at  15000 x g for 1 min 

DISCARD the MB Spin Column

DNA is now ready for downstream applications