Apr 07, 2023
CTAB Extraction Protocol for Sediment
- Cameron R. Turner1
- 1Department of Biological Sciences, University of Notre Dame
Protocol Citation: Cameron R. Turner 2023. CTAB Extraction Protocol for Sediment. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ozkolr2/v1
Manuscript citation:
Turner, C. R., Uy, K. L., & Everhart, R. C. (2015). Fish environmental DNA is more concentrated in aquatic sediments than surface water. Biological Conservation, 183, 93-102. https://doi.org/10.1016/j.biocon.2014.11.017
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
Successfully used by Turner et al. (2015) to detect carp sedDNA
Created: January 13, 2023
Last Modified: April 19, 2023
Protocol Integer ID: 75293
Keywords: Sedimentary DNA, SedDNA, asian carp surface sedimentary dna from experimental pond, ctab extraction protocol for sediment successfully, ctab extraction protocol, sedimentary dna, bigheaded asian carp surface, sediment successfully, extraction, experimental pond, natural river, dna
Abstract
Successfully used by Turner et al., 2015 to detect bigheaded Asian carp surface sedimentary DNA from experimental ponds and natural rivers
Materials
Materials
100 millimolar (mM) Tris-HCL 8
1.4 Mass Percent NaCl
1 Mass / % volume Polyvinylpyrrolidone 8
2 Mass / % volume Cetyl trimethyl ammonium bromide (CTAB)
20 millimolar (mM) EDTA 8
Safety warnings
Steps involving Sevag should be performed inside a fume hood.
Sample preparation
1d 1h 47m 30s
THAW the CTAB-preserved sediment sample in the fridge for no more than 24:00:00
DECONTAMINATE the exterior of the sample tube with 10 % bleach solution and rinse with reverse osmosis water
1d
VORTEX at max speed for 00:00:30
INCUBATE at 60 °C for 00:10:00
10m 30s
Chloroform extraction
1d 1h 47m 30s
ADD 15 mL of Sevag (Chloroform/Isoamyl alcohol 24:1)
Note
Steps involving Sevag should be performed inside a fume hood.
VORTEX the sediment/CTAB/Sevag mixture briefly
SHAKE at low speed (Vortexer setting 4) for 00:05:00
5m
CENTRIFUGE at 3220 x g for 00:15:00 at Room temperature
CAREFULLY transfer the supernatant to a new 50 mL tube
15m
Ethanol precipitation
1d 1h 47m 30s
ADD an equal volume of ice-cold Isopropanol to the supernatant
ADD 1⁄2 volume of 5M NaCl to the supernatant
INCUBATE at -20 °C for 01:00:00 (or overnight if more convenient)
1h
CENTRIFUGE at 3220 x g for 00:15:00 at Room temperature
CAREFULLY pour off the supernatant
15m
ADD 2 mL of 70% EtOH, washing down the inner walls of the tube
CENTRIFUGE at 3220 x g for 00:02:00 at Room temperature
POUR off EtOH and allow the DNA pellet to air dry completely (a 45 °C incubator can be used to evaporate stubborn droplets)
2m
DNA resuspension
1d 1h 47m 30s
RESUSPEND the pellet in 1000 µL of LoTE buffer. Heat briefly at 45 °C and swirl gently to mix and resuspend. Once fully resuspended, briefly centrifuge to collect all liquid in the bottom of 50-mL tube.
Transfer all liquid to a 1.5-μL low-bind microcentrifuge tube
Use 200 µL in OneStepTM Inhibitor Removal Kit (Zymo Research, Irvine, CA)
Protocol references
Turner, C. R., Uy, K. L., & Everhart, R. C. (2015). Fish environmental DNA is more concentrated in aquatic sediments than surface water. Biological Conservation, 183, 93-102. https://doi.org/10.1016/j.biocon.2014.11.017