Modified Organic Extraction Protocol (Pedersen et al., 2016)
Mikkel Pedersen
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
Before start
Review the manufacturer’s Safety Data Sheet and additional chemical information. Ensure that a written experimental protocol including safety information is available.
Steps
Sample preparation & cell lysis
ADD 2g of sediment sample to a sterile spin tube
ADD 170µg of proteinase K to the sample
ADD 3mL of lysis buffer to the sample
VORTEX sample at max speed for 0h 0m 40s
ADD an additional 170µg of proteinase K to each sample
INCUBATE at 37°C
Inhibitor removal
REMOVE PCR inhibitor using MOBIO (MO BIO Laboratories, Carlsbad, CA) C2 and C3 buffers following the manufacturer’s protocol
CENTRIFUGE samples at 10000x g for 0h 3m 0s
TRANSFER supernatant to a sterile microcentrifuge tube
Phenol:Chloroform extraction
ADD equal volume of Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) solution to samples
ROTATE samples gently at Room temperature for 0h 10m 0s
CENTRIFUGE at 3200x g,0h 0m 0sfor 0h 5m 0s
DISCARD supernatant
REPEAT step 4
TRANSFER supernatant to a Millipore 15 mL Amicon Ultra filter for concentration and purification
DNA concentration and purification
CENTRIFUGE filter at 4000x g for 0h 10m 0s
DISCARD liquid flow-through
ADD 500µL EB Buffer (Qiagen) to the filter
CENTRIFUGE at 4000x g for 0h 10m 0s
DISCARD liquid flow-through
REPEAT step 7
ADD 50µL molecular grade water to the filter
INCUBATE at Room temperature for 0h 10m 0s
CENTRIFUGE at 4000x g for 0h 10m 0s
DISCARD filter
DNA is now ready for downstream applications
