TITLE: Environmental DNA (eDNA) Extraction at OSU using the KingFisher Flex with the Omega Bio-Tek Mag-Bind® Blood &amp; Tissue DNA HDQ 96 Kit 
AUTHORS: Jacoby Baker, Kathleen Pitz, Katie Carter

[DESCRIPTION]
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.

[STEPS]
SECTION: MIOP: Minimum Information about an Omics Protocol
1. 
  
MIOP Term
Value
   
methodology category

 
project

 
purpose

 
analyses

 
geographic location

 
broad-scale environmental context

 
local environmental context

 
environmental medium

 
target

 
creator

 
materials required

 
skills required

 
time required

 
personnel required

 
language

 
issued

 
audience

 
publisher

 
hasVersion

 
license

 
maturity level

  

SECTION: AUTHORS
2. 
  
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
   
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SECTION: RELATED PROTOCOLS
3. 
  
PROTOCOL NAME AND LINK
VERSION The version of the protocol you linked to
RELEASE DATE This is the date corresponding to the version listed to the left
   
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SECTION: ACRONYMS AND ABBREVIATIONS
4. 
  
ACRONYM / ABBREVIATION
DEFINITION
   
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SECTION: GLOSSARY
5. 
  
SPECIALISED TERM
DEFINITION
   
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SECTION: EQUIPMENT
13. 
  
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




 
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Consumable equipment




 
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Chemicals




 
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SECTION: STANDARD OPERATING PROCEDURE
14. 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.
SECTION: REFERENCES
45. 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
SECTION: APPENDIX A: DATASHEETS
46. 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.
SECTION: BACKGROUND
6. 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
SECTION: BACKGROUND
7. 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.

SECTION: BACKGROUND
8. 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
SECTION: BACKGROUND
9. Personnel Required

1 Technician
SECTION: BACKGROUND
10. Safety

Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure
SECTION: BACKGROUND
11. Training requirements

Sterile technique, pipetting skills.
SECTION: BACKGROUND
12. Time needed to execute the procedure

Specify how much time is necessary to execute the procedure.
SECTION: PREPARATION
15. 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.
SECTION: EXTRACTION
16. 500
Prepare a mastermix of AL Buffer and Proteinase K Solution only for samples to be
extracted according to the table below:

 
* 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.
SECTION: QUALITY CONTROL
43. Extracted DNA concentrations were Quantified with ThermoFisher Quant-iT dsDNA HS assay on a BioTek Synergy H1 Hybrid Multi-Mode plate reader
SECTION: BASIC TROUBLESHOOTING GUIDE
44. Identify known issues associated with the procedure, if any.

Provide troubleshooting guidelines when available.
SECTION: EXTRACTION
18. Bead beat for 2x45 seconds at 30 Hz on Qiagen Tissue Lyser II
SECTION: EXTRACTION
17. 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
SECTION: EXTRACTION
19.  Incubate overnight at 56C
SECTION: EXTRACTION
24. Add 20 uL HDQ mag beads to one KingFisher Deep Well plate
SECTION: EXTRACTION
23.  Add 320 uL HDQ binding buffer to both KingFisher Deep Well plates
SECTION: EXTRACTION
22. Transfer 200 uL lysate 2x into two KingFisher Deep Well plates filled with 230 uL AL buffer, pipette mix
SECTION: EXTRACTION
21. Spin at 4000 x g for 10 minutes
SECTION: EXTRACTION
20. Add 5 uL RNase A to each sample
SECTION: EXTRACTION
25.2. Repeat steps 25 & 25.1 with the second KingFisher Deep Well plate that did not include the HDQ mag beads
SECTION: EXTRACTION
25. 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.
SECTION: EXTRACTION
26. Remove the plate from the magnetic separation device.
SECTION: EXTRACTION
27. Add 600 μL VHB Buffer.
Note: VHB Buffer must be diluted with 100% ethanol prior to use.
SECTION: EXTRACTION
25.1. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles
HDQ.
SECTION: EXTRACTION
30. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles
HDQ.
SECTION: EXTRACTION
29. 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.
SECTION: EXTRACTION
28. Vortex for 15 seconds to mix.
Note: Complete resuspension of the Mag-Bind‱ Particles HDQ is critical for obtaining
good purity.
SECTION: EXTRACTION
33. Add 600 μL SPM Buffer.
Note: SPM Buffer must be diluted with 100% ethanol prior to use.
SECTION: EXTRACTION
32. Repeat Steps 27-31 for a second VHB Buffer step.
SECTION: EXTRACTION
31. Remove the plate from the magnetic separation device.
SECTION: EXTRACTION
34. Vortex for 15 seconds to mix.
SECTION: EXTRACTION
39. 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.
SECTION: EXTRACTION
37. Select one of the following ethanol removal steps:

SECTION: EXTRACTION
36. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind‱ Particles
HDQ.
SECTION: EXTRACTION
35. 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.
SECTION: EXTRACTION
38. Remove the plate from the magnetic separation device.
SECTION: EXTRACTION
37.1. 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.
SECTION: EXTRACTION
42. Transfer the cleared supernatant containing purified DNA to a 96-well microplate (not
provided). Store DNA at -20°C.
SECTION: EXTRACTION
41. 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.
SECTION: EXTRACTION
40. 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.