Apr 06, 2023

Public workspace[Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Optional Concentration

DOI
dx.doi.org/10.17504/protocols.io.rm7vzb9x4vx1/v1
  • 1Maine Center for Genetics in the Environment, University of Maine
Grayson Huston
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzb9x4vx1/v1
Protocol Citation: Grayson Huston 2023. [Modified] DNeasy PowerSoil Pro Kit_Increased Sediment Volume & Optional Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb9x4vx1/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 collected in a stream 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: 78502
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
Modified PowerSoil Pro extraction - sample preparation & cell lysis
Modified PowerSoil Pro extraction - sample preparation & cell lysis
31m
31m
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 Amount2.0 g of wet sediment to the PowerBead Pro Tube
Note
Qiagen recommends Amount.25 g
In this test, Amount0.25 g , Amount0.5 g , Amount1.0 g , and Amount2.0 g of sediment were tested

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
31m
31m
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
31m
31m
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 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
Modified PowerSoil Pro extraction - wash spin column
31m
31m
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
31m
31m
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
(Optional) DNA Concentration with PALL Nanosep 30K Centrifugal Devices
(Optional) DNA Concentration with PALL Nanosep 30K Centrifugal Devices
2m
2m
ENSURE that the sample reservoir is firmly placed into the filtrate receiver

PIPETTE Amount50-100 µL of DNA extract into the sample reservoir

CAP the Nanosep device
CENTRIFUGE at Amount5000 x g for Duration00:02:00

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
Note
If the sample appears to have "spun dry", recover the sample by pipetting ~ 20uL of elution buffer onto the membrane and recovering with a micropipette

2m