Apr 06, 2023
[Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Inhibitor Removal Pre-Extraction
- 1Maine Center for Genetics in the Environment
Protocol Citation: Grayson Huston 2023. [Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Inhibitor Removal Pre-Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2be9g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
Protocol successful at detecting fish sedDNA from stream sediment collected during a fish migration
Protocol unsuccessful at detecting fish sedDNA from Maine lakes
Created: March 10, 2023
Last Modified: April 06, 2023
Protocol Integer ID: 78512
Keywords: Sedimentary DNA, SedDNA, Fish
Abstract
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
Wash buffer reagents
Wash buffer reagents
CREATE 0.5M EDTA, pH 8.0; final volume: 250mL
Note
- ADD 52.025 g Ethylenediamine Tetraacetic Acid, Tetrasodium Salt Dihydrate (EDTA) to a volumetric flask
- ADD 200 mL DI water to dissolve
- TITRATE to pH 8.0 with Hydrochloric Acid (HCL, approx. 9.5 mL)
- ADD DI water to bring final volume to 250 mL
- AUTOCLAVE solution
CREATE 1M Tris-HCl, pH 8.0; final volume: 500mL
Note
- ADD 78.8 g Tris-HCl to a volumetric flask
- ADD 400 mL DI water to dissolve
- RAISE pH to 8.0 with 10 N NaOH
- ADD DI water to bring final volume to 500 mL
- AUTOCLAVE solution
CREATE a batch of 0.5M Na2PO4*7H2O, pH 8.0; final volume: 250mL
Note
- ADD 33.508 g Na2PO4*7H2O to a volumetric flask
- ADD 200 mL DI water and heat to dissolve
- RAISE pH to 8.0 with 10 N NaOH (approx. 1.5 mL)
- ADD DI water to bring the final volume to 250 mL
- AUTOCLAVE solution
CREATE 10N NaOH; final volume: 40mL
Note
- ADD 16 g NaOH to a volumetric flask
- ADD 40 mL of DI water to dissolve
MIX Inhibitor Removal Wash Buffer; final volume: 500mL
Note
- 10 mL 0.5M EDTA, pH 8.0
- 25 mL 1M Tris-HCl, pH 8.0
- 50 mL Na2PO4*7H2P, pH 8.0
- 415 mL DI water
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
PCR inhibitor removal via wash buffer
PCR inhibitor removal via wash buffer
10m 30s
10m 30s
WEIGH sediment samples into a centrifuge-safe tube
ADD approximately 3x the volume of Wash Buffer to the sediment samples
Note
Account for loss of sediment during wash process (e.g., wash 5g if you plan to extract 3g of sediment)
If you measure 5g sediment, add ~15 mL of Wash Buffer
VORTEX sample at maximum speed for 00:00:30
CENTRIFUGE at 1760 x g for 00:10:00
DISCARD supernatant
10m 30s
REPEAT steps 6 and 7 three to four times
PROCEED with DNA extractions
Modified PowerSoil pro extraction - sample preparation & cell lysis
Modified PowerSoil pro extraction - sample preparation & cell lysis
23m
23m
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
SECURE PowerBead Pro Tubes horizontally to a 1.5mL-2.0mL Vortex Adapter
VORTEX for 00:10:00
ROTATE tubes so caps are oriented in opposite direction
VORTEX for another 00:10:00
20m
CENTRIFUGE PowerBead Pro Tube at 15000 x g for 00:01:00
TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube
1m
Modified PowerSoil pro extraction - inhibitor removal
Modified PowerSoil pro extraction - inhibitor removal
23m
23m
ADD 200 µL of Solution CD2
VORTEX briefly to mix
CENTRIFUGE at 15000 x g for 00:01:00
AVOIDING the pellet, transfer all supernatant to a clean 2 mL Microcentrifuge Tube
1m
Modified PowerSoil pro extraction - bind DNA
Modified PowerSoil pro extraction - bind DNA
23m
23m
ADD 600 µL of Solution CD3
VORTEX briefly to mix
LOAD 650 µL of the lysate onto a MB Spin Column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the liquid flow-through
1m
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
Modified PowerSoil pro extraction - wash spin column
Modified PowerSoil pro extraction - wash spin column
23m
23m
ADD 500 µL of Solution EA to the MB Spin column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the liquid flow-through and place the MB Spin Column into same 2 mL Collection Tube
1m
ADD 500 µL of Solution C5 to the MB Spin Column
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the liquid flow-through and place the MB Spin Column into a new 2 mL Collection Tube
1m
CENTRIFUGE at 16000 x g for 00:02:00
CAREFULLY place the MB Spin Column into a new 1.5mL Elution Tube
2m
Modified PowerSoil pro extraction - elute the DNA
Modified PowerSoil pro extraction - elute the DNA
23m
23m
ADD 100 µL of Solution C6 to the center of the white membrane in the MB Spin Column
INCUBATE at Room temperature for 00:01:00
CENTRIFUGE at 15000 x g for 00:01:00
2m
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 00:01:00
CENTRIFUGE at 15000 x g for 00:01:00
DISCARD the MB Spin Column
DNA is now ready for downstream applications
2m
Protocol references
Wash Buffer from Poulain et al., 2015; modified from Zhou, Bruns and Tiedje., 1996