Apr 05, 2023

Public workspace[Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Inhibitor Removal Pre-Extraction

DOI
dx.doi.org/10.17504/protocols.io.yxmvm2be9g3p/v1
  • 1Maine Center for Genetics in the Environment
Grayson Huston
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm2be9g3p/v1
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 05, 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
  1. ADD Amount52.025 g Ethylenediamine Tetraacetic Acid, Tetrasodium Salt Dihydrate (EDTA) to a volumetric flask
  2. ADD Amount200 mL DI water to dissolve
  3. TITRATE to pH 8.0 with Hydrochloric Acid (HCL, approx. 9.5 mL)
  4. ADD DI water to bring final volume to 250 mL
  5. AUTOCLAVE solution

CREATE 1M Tris-HCl, pH 8.0; final volume: 500mL
Note
  1. ADD Amount78.8 g Tris-HCl to a volumetric flask
  2. ADD Amount400 mL DI water to dissolve
  3. RAISE pH to 8.0 with Amount10 N NaOH
  4. ADD DI water to bring final volume to 500 mL
  5. AUTOCLAVE solution

CREATE a batch of 0.5M Na2PO4*7H2O, pH 8.0; final volume: 250mL

Note
  1. ADD Amount33.508 g Na2PO4*7H2O to a volumetric flask
  2. ADD Amount200 mL DI water and heat to dissolve
  3. RAISE pH to 8.0 with Amount10 N NaOH (approx. 1.5 mL)
  4. ADD DI water to bring the final volume to 250 mL
  5. AUTOCLAVE solution


CREATE 10N NaOH; final volume: 40mL
Note
  1. ADD Amount16 g NaOH to a volumetric flask
  2. ADD Amount40 mL of DI water to dissolve

MIX Inhibitor Removal Wash Buffer; final volume: 500mL
Note
  1. Amount10 mL 0.5M EDTA, pH 8.0
  2. Amount25 mL 1M Tris-HCl, pH 8.0
  3. Amount50 mL Na2PO4*7H2P, pH 8.0
  4. Amount415 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 Duration00:00:30

CENTRIFUGE at Amount1760 x g for Duration00:10:00

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 Amount0.5 g of washed sediment to the PowerBead Pro Tube

ADD Amount800 µL Solution CD1

VORTEX briefly to mix
SECURE PowerBead Pro Tubes horizontally to a 1.5mL-2.0mL Vortex Adapter

VORTEX for Duration00:10:00

ROTATE tubes so caps are oriented in opposite direction

VORTEX for another Duration00:10:00
20m
CENTRIFUGE PowerBead Pro Tube at Amount15000 x g for Duration00:01:00

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 Amount200 µL of Solution CD2

VORTEX briefly to mix
CENTRIFUGE at Amount15000 x g for Duration00:01:00

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 Amount600 µL of Solution CD3

VORTEX briefly to mix
LOAD Amount650 µL of the lysate onto a MB Spin Column

CENTRIFUGE at Amount15000 x g for Duration00:01:00

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 Amount500 µL of Solution EA to the MB Spin column

CENTRIFUGE at Amount15000 x g for Duration00:01:00

DISCARD the liquid flow-through and place the MB Spin Column into same 2 mL Collection Tube
1m
ADD Amount500 µL of Solution C5 to the MB Spin Column

CENTRIFUGE at Amount15000 x g for Duration00:01:00

DISCARD the liquid flow-through and place the MB Spin Column into a new 2 mL Collection Tube
1m
CENTRIFUGE at Amount16000 x g for Duration00:02:00

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 Amount100 µL of Solution C6 to the center of the white membrane in the MB Spin Column

INCUBATE at TemperatureRoom temperature for Duration00:01:00

CENTRIFUGE at Amount15000 x g for Duration00:01:00

2m
PIPETTE the liquid flow-through and re-add it to the center of the white membrane in the MB Spin Column

INCUBATE at TemperatureRoom temperature for Duration00:01:00

CENTRIFUGE at Amount15000 x g for Duration00:01:00

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