Sep 03, 2024

Public workspaceEnvironmental DNA (eDNA) Extraction at OSU using the KingFisher Flex with the Omega Bio-Tek Mag-Bind® Blood & Tissue DNA HDQ 96 Kit

 Forked from DNA EXTRACTION Protocol Template
DOI
dx.doi.org/10.17504/protocols.io.14egn6rxql5d/v1
  • 1MBARI;
  • 2OSU
  • Better Biomolecular Ocean Practices (BeBOP)
Jacoby Baker
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DOI: dx.doi.org/10.17504/protocols.io.14egn6rxql5d/v1
Protocol Citation: Jacoby Baker, Kathleen Pitz, Katie Carter 2024. Environmental DNA (eDNA) Extraction at OSU using the KingFisher Flex with the Omega Bio-Tek Mag-Bind® Blood & Tissue DNA HDQ 96 Kit . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6rxql5d/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: August 20, 2024
Last Modified: September 03, 2024
Protocol Integer ID: 106027
Keywords: DNA Extractions, KingFisher
Abstract
This protocol describes the DNA extraction process at Oregon State University (OSU) using the KingFisher Flex with the Omega Bio-Tek Mag-Bind Blood & Tissue DNA HDQ 96 Kit. Specific modifications to the original protocol are bolded for easy identification.
MIOP: Minimum Information about an Omics Protocol
MIOP: Minimum Information about an Omics Protocol

MIOP TermValue
analysesDNA extraction [OBI:0000257]
audienceScientists
broad-scale environmental contextmarine biome ENVO_00000447
creatorJacoby Baker, https://orcid.org/0000-0002-0673-7535
environmental mediumsea water [ENVO:00002149] | filter paper [OBI:0000151]
geographic locationMonterey Bay [GAZ:00002509]
hasVersion1
issued2024-08-22
languageen
license
local environmental contextoceanic epipelagic zone biome [ENVO:01000033]
materials requiredcentrifuge [OBI:0400106] | incubator [OBI:0000136]]
maturity levelDemonstrated
methodology categorysample extraction and purification
personnel required1
projectMarine Biodiversity Observation Network (MBON)
publisherMonterey Bay Aquarium Research Institute, Chavez Lab
purposeDNA extraction [OBI:0000257]
skills requiredsterile technique | pipetting skills
targetDNA
time required1320
See https://github.com/BeBOP-OBON/miop/blob/main/model/schema/terms.yaml for list and definitions.

AUTHORS
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
OSU TechnicianOregon State University
Jacoby BakerMBARI0000-0002-0673-7535yyyy-mm-dd

RELATED PROTOCOLS
RELATED PROTOCOLS

PROTOCOL NAME AND LINKISSUER / AUTHORRELEASE / ACCESS DATE
Sterile technique, pipetting skills.Omega Bio-Tek2024-01-01
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
ACRONYMS AND ABBREVIATIONS

ACRONYM / ABBREVIATIONDEFINITION
PVDFpolyvinylidene difluoride

GLOSSARY
GLOSSARY

SPECIALISED TERMDEFINITION
Content CellContent Cell
Content CellContent Cell

BACKGROUND
BACKGROUND
Summary

This protocol is a modified version of the Omega Bio-Tek Mag-Bind Blood & Tissue DNA HDQ 96 Kit used for DNA extractions at Oregon State University's Center for Quantitative Life Sciences
Method description and rationale

This modified protocol is used to extract environmental DNA from filtered seawater samples. It has been applied to 0.22μm PVDF and 0.45μm PVDF.

Spatial coverage and environment(s) of relevance
This protocol has been used to extract DNA from filtered sea water samples taken from marine coastal stations.
sea water [ENVO:00002149]
http://purl.obolibrary.org/obo/ENVO_00002149
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.
Time needed to execute the procedure
Specify how much time is necessary to execute the procedure.
EQUIPMENT
EQUIPMENT

DESCRIPTION e.g. filterPRODUCT NAME AND MODEL Provide the official name of the productMANUFACTURER Provide the name of the manufacturer of the product.QUANTITY Provide quantities necessary for one application of the standard operating procedure (e.g. number of filters).REMARK For example, some of the consumable may need to be sterilized, some commercial solution may need to be diluted or shielded from light during the operating procedure.
Durable equipment
Mag-Bind® Blood & Tissue DNA HDQ 96 Kit v8.2Mag-Bind® Blood & Tissue DNA HDQ 96 Kit v8.2Omega Bio-TekContent CellCat # M6399
bead beaterTissueLyser IIQiagenCAT# 85300
Consumable equipment
homogenization plateQiagen collection microtubesQiagen1Content Cell
5 mm stanless steel bashing beadsStainless Steel Beads, 5 mmQiagen96Cat # 69989
KingFisher tip combKingFisher 96 tip comb for deep-well magnets, 10 x 10 pcs/box (for Flex and Presto)1Cat # 97002534
2ml deep well platesKingFisher 96 deep-well plate, sterile (for Duo Prime, Flex and Presto)KingFisherCat # 95040460
96-well Microplatee.g., Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted, white/cleare.g., Bio Rade.g., Cat # HSP9601
Chemicals
TL BufferTL BufferOmega Bio-TekExtra volume needed; Cat # PD061
Proteinase K SolutionProteinase K SolutionOmega Bio-TekExtra volume needed; Cat # PROK-50
Molecular grade Ethanol
NF H2ONuclease Free WaterInvitrogenCAT# AM9932
RNase ARNase AOmega Bio-TekCat # AC118 RNase A

STANDARD OPERATING PROCEDURE
STANDARD OPERATING PROCEDURE
The following protocol is modified from the Mag-Bind‱ Blood & Tissue DNA HDQ 96 Kit Protocol (https://omegabiotek.com/product/tissue-and-blood-kit-genomic-dna-isolation-mag-bind-hdq-96/?cn-reloaded=1).

Modified portions are Bolded.
PREPARATION
PREPARATION
Before Starting: • Prepare SPM Buffer, VHB Buffer, and HDQ Binding Buffer according to the ”Preparing Reagents” section on Page 4 of the Mag-Bind‱ Blood & Tissue DNA HDQ 96 Kit Protocol (https://omegabiotek.com/product/tissue-and-blood-kit-genomic-dna-isolation-mag-bind-hdq-96/?cn-reloaded=1). • Set heat block, incubator, or water bath to 56°C.
EXTRACTION
EXTRACTION
500
Prepare a mastermix of AL Buffer and Proteinase K Solution only for samples to be extracted according to the table below:

ComponentAmount to PrepTotal Amount per 96-well Plate
AL Buffer500 µL52.8 mL*
Proteinase K Solution40 µL4.2 mL*
* 10% excess volume has been calculated for a 96-well plate. Important: Only prepare as much AL Buffer/Proteinase K Solution mastermix that will be used within 4 hours of preparation.
Transfer filters into homogenization plate (Qiagen collection microtubes with 5mm stainless steel bead in each tube) with 500 uL TL buffer and 40 uL ProK
Bead beat for 2x45 seconds at 30 Hz on Qiagen Tissue Lyser II
 Incubate overnight at 56C
Add 5 uL RNase A to each sample
Spin at 4000 x g for 10 minutes
Transfer 200 uL lysate 2x into two KingFisher Deep Well plates filled with 230 uL AL buffer, pipette mix
 Add 320 uL HDQ binding buffer to both KingFisher Deep Well plates
Add 20 uL HDQ mag beads to one KingFisher Deep Well plate
Place the plate on a magnetic separation device to magnetize the Mag-Bind‱ Particles HDQ. Let sit at room temperature until the Mag-Bind‱ Particles HDQ are completely cleared from solution.
Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles HDQ.
Repeat steps 25 & 25.1 with the second KingFisher Deep Well plate that did not include the HDQ mag beads
Remove the plate from the magnetic separation device.
Add 600 μL VHB Buffer.
Note: VHB Buffer must be diluted with 100% ethanol prior to use.
Vortex for 15 seconds to mix. Note: Complete resuspension of the Mag-Bind‱ Particles HDQ is critical for obtaining good purity.
Place the plate on the magnetic separation device to magnetize the Mag-Bind‱ Particles HDQ. Let sit at room temperature until the Mag-Bind‱ Particles HDQ are completely cleared from solution.
Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles HDQ.
Remove the plate from the magnetic separation device.
Repeat Steps 27-31 for a second VHB Buffer step.
Add 600 μL SPM Buffer. Note: SPM Buffer must be diluted with 100% ethanol prior to use.
Vortex for 15 seconds to mix.
Place the plate on the magnetic separation device to magnetize the Mag-Bind‱ Particles HDQ. Let sit at room temperature until the Mag-Bind‱ Particles HDQ are completely cleared from solution.
Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles HDQ.
Select one of the following ethanol removal steps:
A. Leave the plate on the magnetic separation device. Add 500 µL nuclease-free water (not provided), leave on magnet for 20-30 seconds, and then aspirate. Do not leave nuclease-free water on Mag-Bind‱ Particles HDQ for more than 60 seconds. Continue to Step 40. OR
B. Leave the plate on the magnetic separation device. Wait 1 minute. Remove residual liquid with a pipettor. Dry the Mag-Bind‱ Particles HDQ for an additional 10 minutes. Continue to Step 40.
Remove the plate from the magnetic separation device.
Add 100 μL Elution Buffer to elute DNA from the Mag-Bind‱ Particles HDQ. Note: Heat Elution Buffer or nuclease-free water to 70°C to improve yield.
Vortex for 5 minutes to mix. Note: If constant vortexing for 5 minutes is not possible, vortex for 15 seconds every 1-2 minutes for 5 minutes.
Place the plate on the magnetic separation device to magnetize the Mag-Bind‱ Particles HDQ. Let sit at room temperature until the Mag-Bind‱ Particles HDQ are completely cleared from solution.
Transfer the cleared supernatant containing purified DNA to a 96-well microplate (not provided). Store DNA at -20°C.
QUALITY CONTROL
QUALITY CONTROL
Extracted DNA concentrations were Quantified with ThermoFisher Quant-iT dsDNA HS assay on a BioTek Synergy H1 Hybrid Multi-Mode plate reader
BASIC TROUBLESHOOTING GUIDE
BASIC TROUBLESHOOTING GUIDE
Identify known issues associated with the procedure, if any. Provide troubleshooting guidelines when available.
REFERENCES
REFERENCES
This protocol is a modified version of the Omega Bio-Tek Mag-Bind Blood & Tissue DNA HDQ 96 Kit (https://omegabiotek.com/product/tissue-and-blood-kit-genomic-dna-isolation-mag-bind-hdq-96/?cn-reloaded=1) used for DNA extractions at Oregon State University's Center for Quantitative Life Sciences
APPENDIX A: DATASHEETS
APPENDIX A: DATASHEETS
Link templates (e.g. preformatted spreadsheets) used to record measurements and report on the quality of the data as well as any documents such as manufacturer specifications, images, etc that support this protocol. Please include a short note describing the document’s relevance.
Protocol references