Apr 07, 2023

Modified Organic Extraction Protocol (Pedersen et al., 2016)

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1o3oklr2/v1
  • Mikkel Pedersen1
  • 1Centre for GeoGenetics, Natural History Museum, University of Copenhagen
Mark Louie Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1o3oklr2/v1
Protocol Citation: Mikkel Pedersen 2023. Modified Organic Extraction Protocol (Pedersen et al., 2016). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1o3oklr2/v1
Manuscript citation:
Pedersen, M., Ruter, A., Schweger, C. et al. Postglacial viability and colonization in North America’s ice-free corridor. Nature 537, 45–49 (2016). https://doi.org/10.1038/nature19085
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: January 20, 2023
Last Modified: April 19, 2023
Protocol  Integer ID: 75593
Keywords: Sedimentary DNA, SedDNA, version of the organic extraction protocol, organic extraction protocol, modified organic extraction protocol, kya sediment core, extraction, biodiversity
Abstract
A modified version of the organic extraction protocol used in Wales et al., 2014.

Protocol successful at reconstructing the biodiversity of 15 kya sediment core, including a few fish species
Guidelines
Phenol should be handled with care as it is corrosive and toxic.
Chloroform is toxic if swallowed or inhaled. It can cause severe and irreversible health effects, including death
Materials
Lysis buffer
Proteinase K
MO BIO's PowerClean DNA Clean Up Kit
Phenol: Chloroform: Isoamyl alcohol
Ethanol
TE Buffer
30 kDa Amicon Ultra-4 centrifugal filters
Safety warnings
Skin exposure to large amounts of phenol or chloroform can cause sores. Skin exposure to lesser amounts of phenol or chloroform can cause irritation. Phenol and chloroform can irritate theeyes that encounter it.
Before start
Review the manufacturer’s Safety Data Sheet and additional chemical information. Ensure that a written experimental protocol including safety information is available.
Sample preparation & cell lysis
53m 40s
ADD 2 g of sediment sample to a sterile spin tube

ADD 170 µg of proteinase K to the sample

ADD 3 mL of lysis buffer to the sample

VORTEX sample at max speed for 00:00:40
Note
Lysis buffer:
  • 68 millimolar (mM) N-lauroylsarcosine sodium salt
  • 50 millimolar (mM) Tris-HCl (pH 8.0)
  • 150 millimolar (mM) NaCl
  • 20 millimolar (mM) EDTA (pH 8.0) and,

Immediately before extraction, for each 30 mL of lysis buffer add:
  • 1.5 mL 2-mercaptoethanol
  • 1 mL 1 M DTT

40s
ADD an additional 170 µg of proteinase K to each sample

INCUBATE Overnight at 37 °C
Inhibitor removal
53m 40s
REMOVE PCR inhibitor using MOBIO (MO BIO Laboratories, Carlsbad, CA) C2 and C3 buffers following the manufacturer’s protocol

Note

CENTRIFUGE samples at 10000 x g for 00:03:00

TRANSFER supernatant to a sterile microcentrifuge tube

3m
Phenol:Chloroform extraction
53m 40s
ADD equal volume of Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) solution to samples

ROTATE samples gently at Room temperature for 00:10:00

CENTRIFUGE at 3200 x g for 00:05:00

DISCARD supernatant
15m
REPEAT step 4

TRANSFER supernatant to a Millipore 15 mL Amicon Ultra filter for concentration and purification
DNA concentration and purification
40m
CENTRIFUGE filter at 4000 x g for 00:10:00

DISCARD liquid flow-through
10m
ADD 500 µL EB Buffer (Qiagen) to the filter

CENTRIFUGE at 4000 x g for 00:10:00

DISCARD liquid flow-through
10m
REPEAT step 7
ADD 50 µL molecular grade water to the filter

INCUBATE at Room temperature for 00:10:00

CENTRIFUGE at 4000 x g for 00:10:00

DISCARD filter

DNA is now ready for downstream applications

20m
Protocol references
Pedersen, M., Ruter, A., Schweger, C. et al. (2016). Postglacial viability and colonization in North America’s ice-free corridor. Nature 537, 45–49. https://doi.org/10.1038/nature19085

Wales, N., Andersen, K., Cappellini, E., Avila-Arcos, M. C. & Gilbert, M. T. Optimization of DNA recovery and amplification from non-carbonized archaeobotanical remains.PLoS One9, e86827 (2014)