Lakes ABPS Protocol - Optimized protocol for the extraction of fish DNA from freshwater sediments (Thomson-Laing et al., 2022)
Georgia Thomson-Laing
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.
Steps
Alkaline extraction
INTO a 50 mL tube add:
10g of sediment sample
6mL sodium hydroxide (0.33M)
3mL Tris-EDTA (pH 8)
VORTEX for 0h 1m 0s
INCUBATE at 65°C for 0h 50m 0s
ALLOW samples to cool to Room temperature
CENTRIFUGE at 15000x g for 1h 0m 0s
Ethanol precipitation
TRANSFER 7.5mL of supernatant to a new 50 mL tube
ADD 7.5mL of Tris HCl (1M, pH 6.7) to neutralize
ADD 1.5mL sodium acetate (3M, pH 5.2)
ADD 30mL of molecular grade 100% ethanol
INCUBATE samples at -20°C for <1h 0m 0s
CENTRIFUGE samples at 10000x g for 1h 0m 0s
DISCARD supernatant
RETAIN precipitated pellet
DNeasy PowerSoil extraction
EXTRACT the total pellet using multiple DNeasy PowerSoil DNA Isolation Kit extractions following the manufacturer's instructions
POOL resultant DNA elutes
DNA is now ready for downstream applications
