May 30, 2024

Public workspaceDNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform

DOI
dx.doi.org/10.17504/protocols.io.14egn63oql5d/v1
DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform
  • 1Hakai Institute
Andreas Novotny
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn63oql5d/v1
External link: https://hakai.org
Protocol Citation: Colleen Kellogg 2024. DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn63oql5d/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 27, 2024
Last Modified: May 30, 2024
Protocol Integer ID: 100675
Keywords: eDNA, marine microbiology, biodiversity
Abstract
This protocol is used to extract genomic DNA from 0.22μl sterivex filters using Phenol-Chlorophorm. As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. This protocol is developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing.
Guidelines
MIOP: Minimum Information about an Omics Protocol
MIOP TermValue
analysesNucleic Acid Extraction
audiencescientists
broad-scale environmental contextmarine biome ENVO_00000447
creatorColleen Kellogg
environmental mediumsea water [ENVO:00002149]
geographic locationNorth Pacific Ocean [GAZ:00002410]
hasVersion1
issued2017
languageen
licenseCC BY 4.0
local environmental contextcoastal sea water [ENVO: 00002150]
materials requiredSterile workbench, Fume Hood, Centrifuge, Incubator
maturity levelMature
methodology categorySample collection
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

RELATED PROTOCOLS
PROTOCOL NAME AND LINKISSUER / AUTHORRELEASE / ACCESS DATE
Seawater filtrationHakai Institute
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 is used to extract genomic DNA from 0.22μl sterivex filters using Phenol-Chlorophorm.
Spatial coverage and environments of relevance As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015, developing a climatology from which we can begin uncover the physical, chemical and biological drivers of community and functional change in the dynamic coastal waters of coastal British Columbia. This protocol is developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing.
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
Reagent1M TE buffer (1M Tris-HCl, 0.1M EDTA, pH 8.0)
Step 2
ReagentProteinase K, 2mLQiagenCatalog #19131
Step 2
Reagent500ml SDS [20%]G-BiosciencesCatalog #786-016
Step 2
ReagentChloroform:Isoamyl alcohol 24:1Merck MilliporeSigma (Sigma-Aldrich)Catalog #C0549
Step 2
ReagentRNAse AQiagenCatalog #19101
Step 2
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1) pH 8.0Thermo Fisher ScientificCatalog #15593049
Step 2
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Step 2
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Step 2
ReagentAmicon™ Ultra Centrifugal Filter UnitsThermo FisherCatalog #UFC801096
Step 2
Safety warnings
Attention


Screenshot 2024-05-24 at 5.16.05 PM.png
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
The protocol assumes that water has been collected on a sterivex filter and preserved with Sucrose lysis buffer. See the following protocol:
Protocol
Seawater Filtration for Microbial or Environmental DNA
NAME
Seawater Filtration for Microbial or Environmental DNA
CREATED BY
Andreas Novotny


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 follows:
Protocol
Sucrose lysis buffer
NAME
Sucrose lysis buffer
CREATED BY
Andreas Novotny

Toxic
  • Thaw filters on ice, if applicable.
  • Replace Parafilm on each sample if needed.
  • Prepare extraction negative - add 1800 µL of SLB (filter-sterilized and UVed) into a new Sterivex.
  • Seal the bottom with parafilm and the top with cap or parafilm, treat the same as the rest of the samples.
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 filter.
  • Reseal the top with Parafilm or a luer cap.
  • Incubate Sterivex in a rotating incubator at 37°C for 1h (in labeled 50 ml falcon tubes).

Incubation
  • Add 100 µL Proteinase K and 100 µL (20%) SDS to each filter.
  • Reseal using Parafilm or a luer cap.
  • Incubate at 55°C for 1-2 hours in a rotating incubator (in 50ml falcon tubes; be sure to keep the same falcon tube for the same sterivex throughout these incubation steps).

Incubation
  • Remove lysate from sterivex filter using a 10cc syringe into a labeled 15 ml falcon tube, use a new syringe for each sample.
Note
Do not pull up on the plunger while sterivex is attached. You will likely need to do multiple plunges to empty the Sterivex, so unscrew the syringe from the filter, pull back the plunger, reattach to Sterivex, and push and hold to plunge liquid out of Sterivex.

  • Repeat as needed until (nearly) all liquid and foam are out of Sterivex.
  • Add 1mL sucrose lysis buffer (SLB), rotate Sterivex with your hands several times to rinse the filter out, and then plunge out the liquid as described above.
  • Pool this with the lysate in the 15 ml tube.
EXTRACTION & CENTRIFUGATION
EXTRACTION & CENTRIFUGATION

  • 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.

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 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