Apr 07, 2023

Public workspaceCTAB Extraction Protocol for Sediment

DOI
dx.doi.org/10.17504/protocols.io.ewov1ozkolr2/v1
  • Cameron R. Turner1
  • 1Department of Biological Sciences, University of Notre Dame
Yuanyu Cheng
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DOI: dx.doi.org/10.17504/protocols.io.ewov1ozkolr2/v1
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 14, 2023
Last Modified: April 19, 2023
Protocol Integer ID: 75293
Keywords: Sedimentary DNA, SedDNA
Abstract
Successfully used by Turner et al., 2015 to detect bigheaded Asian carp surface sedimentary DNA from experimental ponds and natural rivers

https://www.sciencedirect.com/science/article/pii/S000632071400442X
Materials
Materials Concentration100 millimolar (mM) Tris-HCL Ph8
Concentration1.4 Molarity (M) NaCl
Concentration1 Mass / % volume Polyvinylpyrrolidone Ph8
Concentration2 Mass / % volume Cetyl trimethyl ammonium bromide (CTAB)
Concentration20 millimolar (mM) EDTA Ph8

Safety warnings
Steps involving Sevag should be performed inside a fume hood.
Sample preparation
Sample preparation
1d 1h 47m 30s
1d 1h 47m 30s
THAW the CTAB-preserved sediment sample in the fridge for no more than Duration24:00:00

DECONTAMINATE the exterior of the sample tube with Amount10 % bleach solution and rinse with reverse osmosis water
1d
VORTEX at max speed for Duration00:00:30

INCUBATE at Temperature60 °C for Duration00:10:00
10m 30s
Chloroform extraction
Chloroform extraction
1d 1h 47m 30s
1d 1h 47m 30s
ADD Amount15 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 Duration00:05:00
5m
CENTRIFUGE at Amount3220 x g for Duration00:15:00 at TemperatureRoom temperature

CAREFULLY transfer the supernatant to a new 50 mL tube
15m
Ethanol precipitation
Ethanol precipitation
1d 1h 47m 30s
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 Temperature-20 °C for Duration01:00:00 (or overnight if more convenient)
1h
CENTRIFUGE at Amount3220 x g for Duration00:15:00 at TemperatureRoom temperature

CAREFULLY pour off the supernatant
15m
ADD Amount2 mL of 70% EtOH, washing down the inner walls of the tube

CENTRIFUGE at Amount3220 x g for Duration00:02:00 at TemperatureRoom temperature

POUR off EtOH and allow the DNA pellet to air dry completely (a Temperature45 °C incubator can be used to evaporate stubborn droplets)

2m
DNA resuspension
DNA resuspension
1d 1h 47m 30s
1d 1h 47m 30s
RESUSPEND the pellet in Amount1000 µL of LoTE buffer. Heat briefly at Temperature45 °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 Amount200 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