Apr 05, 2023
[Modified] Lake ABPS Protocol - University of Maine
- 1Maine Center for Genetics in the Environment, University of Maine
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, lake abps protocol, version of the lake abps protocol, detecting fish seddna, fish seddna, river sediments during an anadromous fish sea, lake surface sediment, anadromous fish sea, river sediment, protocol
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
4h 1m
CENTRIFUGE sediment samples at 5250 x g for 00:05:00
DISCARD pore water using a sterile pipette, so only sediment remains
5m
ADD 10 g of wet sediment to a sterile 50 mL tube
ADD 6 mL sodium hydroxide (NaOH, 0.33M) to the sample
ADD 3 mL Tris-EDTA (pH 8.0) to the sample
VORTEX sample at max speed for 00:01:00
INCUBATE sample at 65 °C for 00:55:00
ALLOW samples to cool to Room temperature
56m
CENTRIFUGE samples at 5250 x g for 01:00:00
TRANSFER 7.5 mL of supernatant to a new, sterile 50 mL tube
1h
ADD 7.5 mL Tris-HCl (pH 6.7)
ADD 1.5 mL sodium acetate (3M, pH 5.2)
ADD 30 mL molecular grade ethanol
INCUBATE samples at -20 °C for 01:00:00
1h
CENTRIFUGE sample at 5250 x g for 01:00:00
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
29m
WEIGH the concentrated pellet and split it into multiple 0.5 g replicates
TRANSFER each 0.5 g replicate into a PowerBead Pro Tube
ADD 800 µL of Solution CD1 to each PowerBead Pro Tube
SECURE PowerBead Pro Tubes horizontally to a Vortex Adapter
VORTEX for 00:10:00
ROTATE tubes so caps are oriented in the opposite direction
VORTEX for another 00:10:00
20m
CENTRIFUGE sample at 15000 x g for 00:02:00
TRANSFER all supernatant to a clean 2 mL Microcentrifuge Tube
2m
PowerSoil Pro Extraction on Concentrated Pellet - inhibitor removal
29m
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
PowerSoil Pro Extraction on Concentrated Pellet - bind DNA
29m
ADD 600 µL of Solution CD3
VORTEX briefly to mix
LOAD 650 µL of lysate onto an MB spin column
CENTRIFUGE at 15000 x g for 00:01:00
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
29m
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 the 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 2mL Collection Tube
2m
ADD 50-100 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 15000 x g for 00:01:00
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 ~6 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