Apr 05, 2023

Public workspace[Modified] Lake ABPS Protocol - University of Maine

DOI
dx.doi.org/10.17504/protocols.io.x54v9d5yzg3e/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.x54v9d5yzg3e/v1
Protocol Citation: Grayson Huston 2023. [Modified] Lake ABPS Protocol - University of Maine. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d5yzg3e/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: Working
We use this protocol and it's working
Created: March 09, 2023
Last Modified: April 05, 2023
Protocol Integer ID: 78446
Keywords: Sedimentary DNA, SedDNA, Fish
Abstract
A modified version of the Lake ABPS protocol as described in Thomson-Laing et al. 2022

Protocol successful at detecting fish sedDNA collected from lake surface sediments, as well as river sediments during an anadromous fish sea-run migration
Alkaline Lysis & Ethanol Precipitation
Alkaline Lysis & Ethanol Precipitation
4h 1m
4h 1m
CENTRIFUGE sediment samples at Amount5250 x g for Duration00:05:00

DISCARD pore water using a sterile pipette, so only sediment remains
5m
ADD Amount10 g of wet sediment to a sterile 50 mL tube

ADD Amount6 mL sodium hydroxide (NaOH, 0.33M) to the sample

ADD Amount3 mL Tris-EDTA (pH 8.0) to the sample
VORTEX sample at max speed for Duration00:01:00

INCUBATE sample at Temperature65 °C for Duration00:55:00

ALLOW samples to cool to TemperatureRoom temperature
56m
CENTRIFUGE samples at Amount5250 x g for Duration01:00:00

TRANSFER Amount7.5 mL of supernatant to a new, sterile 50 mL tube
1h
ADD Amount7.5 mL Tris-HCl (pH 6.7)

ADD Amount1.5 mL sodium acetate (3M, pH 5.2)

ADD Amount30 mL molecular grade ethanol
INCUBATE samples at Temperature-20 °C for Duration01:00:00
1h
CENTRIFUGE sample at Amount5250 x g for Duration01:00:00

DISCARD supernatant

ALLOW remaining ethanol to evaporate off of concentrated pellet before proceeding to next step
1h
PowerSoil Pro Extraction on Concentrated Pellet - sample preparation & cell lysis
PowerSoil Pro Extraction on Concentrated Pellet - sample preparation & cell lysis
29m
29m
WEIGH the concentrated pellet and split it into multiple Amount0.5 g replicates

TRANSFER each Amount0.5 g replicate into a PowerBead Pro Tube
ADD Amount800 µL of Solution CD1 to each PowerBead Pro Tube

SECURE PowerBead Pro Tubes horizontally to a Vortex Adapter

VORTEX for Duration00:10:00

ROTATE tubes so caps are oriented in the opposite direction

VORTEX for another Duration00:10:00
20m
CENTRIFUGE sample at Amount15000 x g for Duration00:02:00

TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube
2m
PowerSoil Pro Extraction on Concentrated Pellet - inhibitor removal
PowerSoil Pro Extraction on Concentrated Pellet - inhibitor removal
29m
29m
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
PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
29m
29m
ADD Amount600 µL of Solution CD3

VORTEX briefly to mix
LOAD Amount650 µL of lysate onto an MB spin column

CENTRIFUGE at Amount15000 x g for Duration00:01:00

DISCARD the liquid flow-through
1m
REPEAT step 14 to ensure all the lysate has passed through the MB Spin Column

CAREFULLY place the MB spin column into a clean 2mL collection tube
PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
PowerSoil Pro Extraction on Concentrated Pellet - wash spin column
29m
29m
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 the 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 2mL Collection Tube

2m
ADD Amount50-100 µL of Solution C6 to the center of the white membrane in the MB Spin Column
Note
Adjust the amount of Solution C6 added to each replicate so that the final volume, once all replicates are pooled together (step 21), totals 200ul

For example, if at Step 8 sample A weighed 1.0g and was split into two 0.5g replicates: A1 and A2. At this step (Step 19), A1 and A2 would each receive 100ul Solution C6, so that when they are pooled together, their total volume is 200ul

If sample A was split into three 0.5g replicates (A1, A2, and A3), each would receive approximately 66ul of Solution C6

CENTRIFUGE at Amount15000 x g for Duration00:01:00

DISCARD the MB Spin Column

POOL all replicates into a sterile 1.5 mL Microcentrifuge Tube

DNA is now ready for downstream applications
Note
For best results in qPCR, use ~Amount6 µL of extracted DNA template per PCR reaction


1m
Protocol references
Thomson-Laing, G., Howarth, J.D., Vandergoes, M.J., & Wood, S.A. (2022). Optimised protocol for the extraction of fish DNA from freshwater sediments. Freshwater Biology, 67, 1584-1603. https://doi.org/10.1111/fwb.13962