Apr 17, 2023
Lakes ABPS Protocol - Optimized protocol for the extraction of fish DNA from freshwater sediments (Thomson-Laing et al., 2022)
- Georgia Thomson-Laing1
- 1Cawthron Institute, Nelson, New Zealand
Protocol Citation: Georgia Thomson-Laing 2023. Lakes ABPS Protocol - Optimized protocol for the extraction of fish DNA from freshwater sediments (Thomson-Laing et al., 2022). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm24d6g3p/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: April 17, 2023
Last Modified: April 18, 2023
Protocol Integer ID: 80636
Keywords: lakes abps protocol, lake sediment samples by an alkaline lysis method, fish dna from freshwater sediment, fish seddna from surface sediment, comparison of multiple seddna extraction method, lake sediment, range of lake sediment, multiple seddna extraction method, freshwater sediment, detecting fish seddna, protocol for the extraction, fish dna, sediment sample, surface sediment, abps protocol, optimized abps protocol, alkaline buffer, highest concentrations of target genet, extraction, alkaline lysis method, lake, sediment, multiple powersoil extraction, dna, abp
Abstract
DNA was extracted from lake sediment samples by an alkaline lysis method with ethanol precipitation adapted from method described by Kuwae et al. (2020); Sakata et al. (2020b); Sakata et al. (2020a).
In a comparison of multiple sedDNA extraction methods, the ABPS (Alkaline buffer - power soil) protocol yielded the highest concentrations of target genets across a range of lake sediments. This protocol was further optimized (65C incubation temperature, pooling of multiple PowerSoil extractions) to overcome technical challenges related to co-precipitation of organic content in lake-surface sediments.
The optimized ABPS protocol is called the "Lakes ABPS protocol"
This protocol has proven to be successful at detecting fish sedDNA from surface sediments in multiple systems for multiple species.
Alkaline extraction
1h 51m
INTO a 50 mL tube add:
10 g of sediment sample
6 mL sodium hydroxide (0.33M)
3 mL Tris-EDTA (pH 8)
VORTEX for 00:01:00
INCUBATE at 65 °C for 00:50:00
51m
ALLOW samples to cool to Room temperature
CENTRIFUGE at 15000 x g for 01:00:00
1h
Ethanol precipitation
2h
TRANSFER 7.5 mL of supernatant to a new 50 mL tube
ADD 7.5 mL of Tris HCl (1M, pH 6.7) to neutralize
ADD 1.5 mL sodium acetate (3M, pH 5.2)
ADD 30 mL of molecular grade 100% ethanol
INCUBATE samples at -20 °C for <01:00:00
1h
CENTRIFUGE samples at 10000 x g for 01:00:00
DISCARD supernatant
RETAIN precipitated pellet
1h
DNeasy PowerSoil extraction
EXTRACT the total pellet using multiple DNeasy PowerSoil DNA Isolation Kit extractions following the manufacturer's instructions
Note
0.25-0.5 g of pellet per extraction
POOL resultant DNA elutes
DNA is now ready for downstream applications
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