Apr 07, 2023
Modified Organic Extraction Protocol (Pedersen et al., 2016)
- Mikkel Pedersen1
- 1Centre for GeoGenetics, Natural History Museum, University of Copenhagen
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 19, 2023
Last Modified: April 18, 2023
Protocol Integer ID: 75593
Keywords: Sedimentary DNA, SedDNA
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
Sample preparation & cell lysis
53m 40s
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
Inhibitor removal
53m 40s
53m 40s
REMOVE PCR inhibitor using MOBIO (MO BIO Laboratories, Carlsbad, CA) C2 and C3 buffers following the manufacturer’s protocol
Note
Link to MO BIO's PowerClean DNA Clean Up Kit Handbook: https://www.qiagen.com/us/resources/resourcedetail?id=a757e687-7b7a-4801-96bb-6267620414de&lang=en
CENTRIFUGE samples at 10000 x g for 00:03:00
TRANSFER supernatant to a sterile microcentrifuge tube
3m
Phenol:Chloroform extraction
Phenol:Chloroform extraction
53m 40s
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
DNA concentration and purification
40m
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)