Sep 03, 2024
Environmental 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
- Jacoby Baker1,
- Kathleen Pitz1,
- Katie Carter2
- 1MBARI;
- 2OSU
- Better Biomolecular Ocean Practices (BeBOP)
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 21, 2024
Last Modified: September 03, 2024
Protocol Integer ID: 106027
Keywords: DNA Extractions, KingFisher, dna extraction process at oregon state university, dna extraction, tissue dna hdq 96 kit, dna extraction process, tissue dna hdq, kingfisher flex with the omega bio, omega bio, dna, extraction at osu, bind blood, kingfisher flex, edna, extraction process, extraction, environmental dna, bio
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 Term | Value | |
| analyses | DNA extraction [OBI:0000257] | |
| audience | Scientists | |
| broad-scale environmental context | marine biome ENVO_00000447 | |
| creator | Jacoby Baker, https://orcid.org/0000-0002-0673-7535 | |
| environmental medium | sea water [ENVO:00002149] | filter paper [OBI:0000151] | |
| geographic location | Monterey Bay [GAZ:00002509] | |
| hasVersion | 1 | |
| issued | 2024-08-22 | |
| language | en | |
| license | ||
| local environmental context | oceanic epipelagic zone biome [ENVO:01000033] | |
| materials required | centrifuge [OBI:0400106] | incubator [OBI:0000136]] | |
| maturity level | Demonstrated | |
| methodology category | sample extraction and purification | |
| personnel required | 1 | |
| project | Marine Biodiversity Observation Network (MBON) | |
| publisher | Monterey Bay Aquarium Research Institute, Chavez Lab | |
| purpose | DNA extraction [OBI:0000257] | |
| skills required | sterile technique | pipetting skills | |
| target | DNA | |
| time required | 1320 |
See https://github.com/BeBOP-OBON/miop/blob/main/model/schema/terms.yaml for list and definitions.
AUTHORS
| PREPARED BY All authors known to have contributed to the preparation of this protocol, including those who filled in the template. | AFFILIATION | ORCID (visit https://orcid.org/ to register) | DATE | |
| OSU Technician | Oregon State University | |||
| Jacoby Baker | MBARI | 0000-0002-0673-7535 | yyyy-mm-dd |
RELATED PROTOCOLS
| PROTOCOL NAME AND LINK | ISSUER / AUTHOR | RELEASE / ACCESS DATE | |
| Sterile technique, pipetting skills. | Omega Bio-Tek | 2024-01-01 |
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
| ACRONYM / ABBREVIATION | DEFINITION | |
| PVDF | polyvinylidene difluoride |
GLOSSARY
| SPECIALISED TERM | DEFINITION | |
| Content Cell | Content Cell | |
| Content Cell | Content Cell |
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]
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
| DESCRIPTION e.g. filter | PRODUCT NAME AND MODEL Provide the official name of the product | MANUFACTURER 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 v8.2 | Mag-Bind® Blood & Tissue DNA HDQ 96 Kit v8.2 | Omega Bio-Tek | Content Cell | Cat # M6399 | |
| bead beater | TissueLyser II | Qiagen | CAT# 85300 | ||
| Consumable equipment | |||||
| homogenization plate | Qiagen collection microtubes | Qiagen | 1 | Content Cell | |
| 5 mm stanless steel bashing beads | Stainless Steel Beads, 5 mm | Qiagen | 96 | Cat # 69989 | |
| KingFisher tip comb | KingFisher 96 tip comb for deep-well magnets, 10 x 10 pcs/box (for Flex and Presto) | 1 | Cat # 97002534 | ||
| 2ml deep well plates | KingFisher 96 deep-well plate, sterile (for Duo Prime, Flex and Presto) | KingFisher | Cat # 95040460 | ||
| 96-well Microplate | e.g., Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted, white/clear | e.g., Bio Rad | e.g., Cat # HSP9601 | ||
| Chemicals | |||||
| TL Buffer | TL Buffer | Omega Bio-Tek | Extra volume needed; Cat # PD061 | ||
| Proteinase K Solution | Proteinase K Solution | Omega Bio-Tek | Extra volume needed; Cat # PROK-50 | ||
| Molecular grade Ethanol | |||||
| NF H2O | Nuclease Free Water | Invitrogen | CAT# AM9932 | ||
| RNase A | RNase A | Omega Bio-Tek | Cat # AC118 RNase A |
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
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
500
Prepare a mastermix of AL Buffer and Proteinase K Solution only for samples to be
extracted according to the table below:
| Component | Amount to Prep | Total Amount per 96-well Plate | |
| AL Buffer | 500 µL | 52.8 mL* | |
| Proteinase K Solution | 40 µL | 4.2 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
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
Identify known issues associated with the procedure, if any.
Provide troubleshooting guidelines when available.
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
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.