Jun 01, 2024

Public workspaceDNA Extraction from Ethanol Zooplankton Samples - Phenol-Chloroform

DOI
dx.doi.org/10.17504/protocols.io.n92ld88mnv5b/v1
DNA Extraction from Ethanol Zooplankton Samples - Phenol-Chloroform
  • 1University of British Columbia;
  • 2Hakai Institute
Andreas Novotny
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ld88mnv5b/v1
External link: https://hakai.org
Protocol Citation: Andreas Novotny, Colleen Kellogg, rute.carvalho Carvalho, Matt Lemay 2024. DNA Extraction from Ethanol Zooplankton Samples - Phenol-Chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld88mnv5b/v1
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: May 30, 2024
Last Modified: June 01, 2024
Protocol Integer ID: 100959
Keywords: Zooplankton, Biodiversity, DNA
Abstract
This protocol extracts genomic DNA from bulk zooplankton samples, preserved either in ethanol or frozen - using Phenol/Chloroform extraction. The protocol was developed to be as similar as possible to the standard protocol used for environmental DNA at Hakai Institute Ocean Observing Program, to be used for downstream DNA metabarcoding:
CITATION
Colleen Kellogg. DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform. protocols.io.
LINK
https://protocols.io/view/dna-extraction-from-0-22-m-sterivex-filters-phenol-dejb3cin

Guidelines
MIOP: Minimum Information about an Omics Protocol
MIOP TermValue
analysesNucleic Acid Extraction
audiencescientists
broad-scale environmental contextmarine biome ENVO_00000447
creatorAndreas Novotny
environmental mediumsea water [ENVO:00002149]
geographic locationNorth Pacific Ocean [GAZ:00002410]
hasVersion1
issued2024
languageen
licenseCC BY 4.0
local environmental contextZooplankton
materials requiredSterile workbench, Fume Hood, Centrifuge, Incubator
maturity levelMature
methodology categoryDNA extraction
personnel required1
projectBiomolecular surveys of marine biodiversity in the Northern Salish Sea, BC
publisherHakai Institute, Ocean Observing Program
purposeDNA Extraction
skills requiredsterile technique | pipetting skills
targetDNA
time required1 day
AUTHORS
PREPARED BY All authors known to have contributed to the preparation of this protocol, including those who filled in the template.AFFILIATIONORCID (visit https://orcid.org/ to register)DATE
Colleen KelloggHakai Institutehttps://orcid.org/0000-0003-4048-53162017
Andreas NovotnyUniversity of British Columbiahttps://orcid.org/0000-0001-8910-61832024

RELATED PROTOCOLS
PROTOCOL NAME AND LINKISSUER / AUTHORRELEASE / ACCESS DATE
DNA extraction from 0.22um sterivex filters using phenol chloroform.Hakai Institutehttps://dx.doi.org/10.17504/protocols.io.14egn63oql5d/v1
This is a list of other protocols which should be known to users of this protocol. Please include the link to each related protocol.
ACRONYMS AND ABBREVIATIONS
ACRONYM / ABBREVIATIONDEFINITION
GLOSSARY
SPECIALISED TERMDEFINITION

BACKGROUND This protocol extracts genomic DNA from bulk zooplankton samples, preserved either in ethanol or frozen - using Phenol/Chloroform extraction. The protocol was developed to be as similar as possible to the standard protocol used for environmental DNA at Hakai Institute Ocean Observing Program, to be used for downstream DNA metabarcoding:
CITATION
Colleen Kellogg. DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform. protocols.io.
LINK
https://protocols.io/view/dna-extraction-from-0-22-m-sterivex-filters-phenol-dejb3cin
Spatial coverage and environments of relevance As part of the Hakai Institute Ocean Observing Program, zooplankton samples have been collected monthly to biweekly, using a 250um bongo net with an opening diameter of 0.5m, towed from 230m-0m. The zooplankton samples have been preserved in formalin for microscopic analysis and in 99% ethanol for genetic analysis.
Personnel Required 1 Technician
Safety Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure!
Training requirements Sterile technique, pipetting skills. Work-safe laboratory practices.
Time needed to execute the procedure
1 Day (for 24 samples).
Protocol materials
ReagentStainless Steel Beads 0.9 – 2.0 mm blendNext AdvanceCatalog #SSB14B
Step 1
ReagentProteinase K, 2mLQiagenCatalog #19131
Step 1
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Step 1
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1) pH 8.0Thermo Fisher ScientificCatalog #15593049
Step 1
Reagent1M TE buffer (1M Tris-HCl, 0.1M EDTA, pH 8.0)
Step 1
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Step 1
Reagent500ml SDS [20%]G-BiosciencesCatalog #786-016
Step 1
ReagentRNAse AQiagenCatalog #19101
Step 1
ReagentAmicon™ Ultra Centrifugal Filter UnitsThermo FisherCatalog #UFC801096
Step 1
ReagentStainless Steel UFO Beads 3.5 mmNext AdvanceCatalog #SSUFO35
Step 1
ReagentChloroform:Isoamyl alcohol 24:1Merck MilliporeSigma (Sigma-Aldrich)Catalog #C0549
Step 1
Safety warnings
Attention


Several of the chemicals are harmful to humans and the environment. Make a proper risk assessment before starting this procedure. Fumes from the extraction are toxic and waste must be treated with extreme care!
Several of the chemicals are harmful to humans and the environment. Make a proper risk assessment before starting this procedure. Fumes from the extraction are toxic and waste must be treated with extreme care!

Before start
Read Minimum Information about an Omics Protocol (MIOP) and other recommendations under the "Guidelines" tab.
PREPARATIONS
PREPARATIONS

Safety information
Several of the chemicals are harmful to humans and the environment. Make a proper risk assessment before starting this procedure. Fumes from the extraction are toxic and waste must be treated with extreme care!

Materials needed (OR similar):
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
ReagentRNAse AQiagenCatalog #19101
Reagent1M TE buffer (1M Tris-HCl, 0.1M EDTA, pH 8.0)Contributed by users
ReagentProteinase K, 2mLQiagenCatalog #19131
Reagent500ml SDS [20%]G-BiosciencesCatalog #786-016
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1) pH 8.0Thermo Fisher ScientificCatalog #15593049
ReagentChloroform:Isoamyl alcohol 24:1Merck MilliporeSigma (Sigma-Aldrich)Catalog #C0549
ReagentAmicon™ Ultra Centrifugal Filter UnitsThermo FisherCatalog #UFC801096
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Sucrose Lysis Buffer (SLB) is prepared as follow:
Protocol
Sucrose lysis buffer
NAME
Sucrose lysis buffer
CREATED BY
Andreas Novotny
To be Autoclaved:
ReagentStainless Steel UFO Beads 3.5 mmNext AdvanceCatalog #SSUFO35
ReagentStainless Steel Beads 0.9 – 2.0 mm blendNext AdvanceCatalog #SSB14B
Toxic
ZOOPLANKTON HOMOGENIZATION
ZOOPLANKTON HOMOGENIZATION
Subset the bulk zooplankton sample to a to a reasoable volume (here, 1/8th of the original sample):
  • Use a plankton net filter to remove the ethanol preservative (keep the preservative).
  • Resuspend zooplankton in dH2O
  • Use a Fulton splitter to subset 1/8 fraction of the zooplankton sample and add to a 50ml falcon tube.
  • Return the remaining fractions to the ethanol preservative.
  • Centrifuge the falcon tube 5000xg for 5 min to pellet the zooplankton
  • Use a pipett to remove all liquid.
  • Add SLB to the falcon tube, to a total volume of 6mL, and resuspend.
  • Add 3 large UFO beads (3.5 mm) to the falcon tube.
  • Add ~30 small stainless steal beads (0.9-2.0 mm) to the falcon tube.
  • Vortex vigorously at full speed for at least 10 min, or until no visible structures of the zooplankton are left.
  • For each sample, add 1800 µL SLB to new labeled 15mL falcon tubes.
  • Add an aliquot of 100 µL of the homogenized zooplankton sample to the SLB tube. The total volume should be around 1900 µL.
LYSIS AND INCUBATION
LYSIS AND INCUBATION
  • Add 100 µL lysozyme (125 mg fully dissolved in 1000 μl 1 x TE) and 20µL RNase A (10 µg/ml: 1µL in 999µL 1 x TE) to each sample tube.
  • Incubate samples in a rotating incubator at 37°C for 1h.

Incubation
  • Add 100 µL Proteinase K and 100 µL (20%) SDS to each lysate tube.
  • Incubate at 55°C for 1-2 hours in a rotating incubator.

Incubation
Add another 1mL sucrose lysis buffer (SLB) to the lysate tube.
EXTRACTION & CENTRIFUGATION
EXTRACTION & CENTRIFUGATION

Safety information
The following section should be done in a fume hood.

  • In the fume hood, add an equal volume (about 3mL) of Phenol:Chloroform:IAA (25:24:1), pH 8.0 to the lysate tube.
  • Invert for 10 secondsby hand to mix.
  • Spin at 2500 g for 6 min or until the aqueous layer is clear. Wait at least 10 minutes before opening the centrifuge.
  • Transfer the aqueous (top) layer into a new 15 ml falcon tube.
Toxic
  • Add an equal volume (approx 3mL) of Chloroform:IAA (24:1) to the tube containing the aqueous layer.
  • Invert for 10 seconds by hand.
  • Spin at 2500 g for 6 min or until the aqueous layer is clear with no debris. Wait at least 10 minutes before opening the centrifuge.
  • Transfer the aqueous layer into a labeled Amicon Ultra centrifuge tube (UFC801096, EMD Millipore).
  • Top up Amicon with 1-2mL of 1 x TE buffer.
  • Spin at 3500 g for 10-15 minutes.
  • Check to make sure there is less than 1ml liquid left in Amicon at the end of this (if not, fill up with 2mL 1 x TE and spin again).
  • Add 2 mL TE buffer to Amicon and spin at 3500 g for 6 min.
  • Remove filtrate.
  • Repeat Step 8 twice more (total of 3 washes with 2mL TE).
  • For the last wash, spin until 200 – 500 μl remain in Amicon (typically 8-10 minutes).
  • Note the final volume and transfer to a labeled 1.5 μl Eppendorf tube.
  • Rinse Amicon sides with 50 µL of 1xTE and pool with the rest of the sample in a labeled 1.5mL Eppendorf tube.
DNA QUANTIFICATION AND STORING
DNA QUANTIFICATION AND STORING
  • If desired, aliquot 50 µL from the final sample volume into a 1.5ml Eppendorf tube to use as working stock and place in a -20°C freezer.
  • Place the remaining DNA stock in the -80°C freezer for long-term storage.

Quantify DNA stock using Qubit (following manufacturer’s instructions). Use 2 µL of stock DNA when quantifying.

DNA NORMALISATION
DNA NORMALISATION

Note
Optional, but does tend to yield good success during amplification

Make a normalized 2.5 ng/μl DNA stock for PCR :
Calculate the volume of DNA for 2.5 ng/μL stock (there is a spreadsheet for this)
Where [DNA] is the DNA concentration from Qubit and x is the number of uL of the DNA stock that you need to add for a 25 uL volume of a 2.5 ng/uL DNA stock.
Where [DNA] is the DNA concentration from Qubit and x is the number of uL of the DNA stock that you need to add for a 25 uL volume of a 2.5 ng/uL DNA stock.

Calculate the volume of water for 2.5 ng/μL stock


  • Place the 2.5 ng/μL DNA stock in the -20°C freezer.

Protocol references
This protocol is modified from
Colleen Kellogg. DNA Extraction from 0.22 micrometer Sterivex filters - Phenol Chloroform. Protocols.io:
https://protocols.io/view/dna-extraction-from-0-22-m-sterivex-filters-phenol-dejb3cin
....which is a modified version of:
DNA extraction from 0.22 microM Sterivex filters and cesium chloride density gradient centrifugation. Jody J. Wright, Elena Zaikova, David A. Walsh and Steven J. Hallam
https://doi.org/10.3791/1352
Citations
Colleen Kellogg. DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform
https://protocols.io/view/dna-extraction-from-0-22-m-sterivex-filters-phenol-dejb3cin
Colleen Kellogg. DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform
https://protocols.io/view/dna-extraction-from-0-22-m-sterivex-filters-phenol-dejb3cin