Aug 01, 2023
DNA Extraction Protocol from Sterivex Filters
- Meghan M. Shea1,
- Alexandria Boehm1
- 1Stanford University
Protocol Citation: Meghan M. Shea, Alexandria Boehm 2023. DNA Extraction Protocol from Sterivex Filters. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qyyygr2/v1
Manuscript citation:
Shea, M. M., & Boehm, A. B. (2024). Environmental DNA metabarcoding differentiates between micro-habitats within the rocky intertidal. Environmental DNA, 6(2), e521. https://doi.org/10.1002/edn3.521
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: June 16, 2023
Last Modified: August 01, 2023
Protocol Integer ID: 83545
Keywords: environmental DNA, DNA extraction, Sterivex, Qiagen DNeasy Blood and Tissue Kit, dna extraction protocol from sterivex filter, dna from sterivex filter, dna extraction protocol, sterivex filter, extracting dna, protocol originates with environmental dna sample, capped sterivex filter, environmental dna sample, µm capped sterivex filter, environmental dna, filtration protocol, dna, adjusted procedure with the qiagen dneasy blood, extraction, qiagen dneasy blood, filtration, tissue kit
Abstract
This is a modified protocol for extracting DNA from Sterivex filters using an adjusted procedure with the Qiagen DNeasy Blood and Tissue Kit, as first published by Spens et al. 2017.
This protocol originates with environmental DNA samples collected onto 0.22 µm capped Sterivex filters, e.g. through this sampling and filtration protocol:
Protocol
CREATED BY
Meghan M. Shea
Attachments
Image Attribution
Meghan M. Shea
Materials
General Laboratory Equipment:
| Equipment | Specific Model Used | |
| Incubator | VWR 1565B | |
| Tube roller shaker | Southwest Science (STL100) | |
| 10% bleach solution in spray bottle | NA | |
| >70% ethanol solution in spray bottle | NA | |
| RNase Away solution in spray bottle | NA | |
| UV Crosslinker | UVP CL-1000 Ultraviolet Crosslinker | |
| Kimwipes | NA | |
| Gloves | NA | |
| 1000 µL pipette with sterile tips (that fit the top of the Sterivex--ideally long & skinny) | Various | |
| 200 µL pipette with sterile tips | Various | |
| 100 µL pipette with sterile tips | Various | |
| 10 µL pipette with sterile tips | Various | |
| Vortex | VWR Mini Vortexer | |
| Centrifuge | Eppendorf Centrifuge 5424 | |
| Lab markers | VWR (52877-310): https://us.vwr.com/store/product/4597364/vwr-chemical-resistant-laboratory-marker | |
| Cryo-labels | USA Scientific (9187-0100): https://www.usascientific.com/cryo-tags-combo-sheets/p/9187-0100 | |
| Feezer boxes | Cole-Parmer, via Fisher Scientific (3391550): https://www.fishersci.com/shop/products/polarsafe-81-place-polypropylene-storage-boxes-6/03391550 | |
| Variety of sterile or sterilizable tubes for holding aliquots of reagents | Various, including some that fit in heat block | |
| Heat block | VWR Standard Heatblock | |
| Qubit Fluorometer | Invitrogen Qubit 2.0 Fluorometer |
Note
There are many types of incubators and shakers (some combined) that could be used to heat and agitate the Sterivex filters overnight. The Southwest Rock & Roll Lab Tube Roller was by far the most cost effective option we found, and it fit (just barely) in an existing incubator.
Additional Materials:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| Qubit dsDNA Broad Range assay kit | 1 reaction/sample + additional for standards | Fisher Scientific (Q32850) | https://www.fishersci.com/shop/products/qubit-dsdna-hs-br-assay-kits/Q32850 | $115/100 reactions | |
| Sterile 3 mL luer lock syringes | 1/sample | BD, via Fisher Scientific (14-823-435) | https://www.fishersci.com/shop/products/bd-disposable-syringes-luer-lok-tips-3/14823435 | $21.60/200 syringes | |
| Water, DNA Grade, DNASE, Protease free | Variable | Fisher Scientific (BP24701) | https://www.fishersci.com/shop/products/water-dna-grade-dnase-protease-free-fisher-bioreagents/BP24701 | $30.05/1000 mL | |
| 1.5/2 mL LoBind Eppendorf tubes (either size works; prefer 1.5 mL for storage) | 3/sample | USA Scientific (4043-1021/4043-1048) | https://www.usascientific.com/dna-lobind-microcentrifuge-tubes/p/DNA-LB-Micro-Tubes | $38.95/250 tubes | |
| Ethanol, Absolute (200 Proof), Molecular Biology Grade | Variable | Fisher Scientific (BP2818500) | https://www.fishersci.com/shop/products/ethanol-absolute-200-proof-molecular-biology-grade-fisher-bioreagents-3/BP2818500 | $61.77/500 mL | |
| 5 mL LoBind Eppendorf Tubes | 1/sample | USA Scientific (4011-8310) | https://www.usascientific.com/eppendorf-tube-5-0-ml-dna-lobind/p/4011-8310 | $68.5/200 tubes | |
| Qiagen DNeasy Blood & Tissue Kit | 1 reaction/sample | Qiagen (69506) | https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/dneasy-blood-and-tissue-kit?catno=69506 | $692.3/250 preparations | |
| Qubit tubes | 1/sample + additional for standards | Unknown | NA | Unknown |
Note
Due to the modifications to the Qiagen DNeasy Blood & Tissue Kit, some reagents are used in greater volumes than the manufacturer's protocol, and will run out before the kit is completed. Be prepared to order extra Proteinase K, Buffer ATL, and Buffer AL as needed.
Troubleshooting
Safety warnings
Taken directly from the Qiagen DNeasy Blood & Tissue Handbook:
HB-2061-003_HB_DNY_Blood_Tissue_0720_WW.pdf When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit and kit component.
Safety information
Caution: DO NOT add bleach or acidic solutions directly to the sample preparation waste.
Buffers AL and AW1 contain guanidine salts, which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with a suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Day 1 - DNA Lysing
Clean bench area with bleach, ethanol, and RNase away
Turn on incubator and set to 56°C
Wipe down 1000 µL and 200 µL pipettes with RNase Away and UV for 15 minutes on each side
Assemble materials and reagents needed:
- 1000 µL pipette tips (that fit in the inlet of the Sterivex)
- 200 µL pipette tips
- Proteinase K (from Qiagen DNeasy Blood & Tissue Kit)
- ATL Buffer (from Qiagen DNeasy Blood & Tissue Kit)
Remove sterivex filters from -15°C freezer
Note
The roller shaker used (see MATERIALS) only fits 15 Sterivex filters, which creates a 16 sample extraction batch with an additional extraction blank added on Day 2. 16 samples could be well-balanced in the centrifuge used, leading to easier subsequent processing.
If processing a different number of filters, consider the subsequent processing steps (especially centrifuging) when deciding how many to extract at once.
For each filter:
Remove Sterivex from Whirl-Pak bag and remove cap from inlet end of Sterivex filter
Slowly pipette 80 µL of Proteinase K directly on top of the filter through the inlet, avoiding backsplash by expelling slowly
Slowly pipette 720 µL of ATL buffer directly on top of the filter through the inlet, again avoiding backsplash
Secure Sterivex with same luer cap
Handshake vigorously for several seconds
Place all filters onto roller shaker in incubator
Sterivex arranged on roller shaker in incubator (Photo Credit: Meghan M. Shea)
Incubate at 56°C for ~24 hours (minimum of 12 hours; try to incubate for the same amount of time for all filters for a particular project) while rotating at approximately 6 rpm
Day 2 - DNA Extraction
Clean bench area (including vortex), centrifuge area, and centrifuge with bleach, ethanol, and RNase away.
Soak all tube racks in a 10% bleach solution, followed by 3 rinses with DI water. Let dry and UV for 15 minutes
UV Qubit and LoBind tubes for 15 minutes (in pre-sterilized tube racks) and label with sample numbers:
- Qubit: (# of samples+ extraction blank) + 2 for standards
- 1.5/2 mL LoBind: (# of samples + extraction blank) * 3
- 5 mL LoBind: # of samples + extraction blank
- Enough tubes (any type, does not need to be LoBind) to hold AE Buffer that fit in heat block (see Step 15), and to hold Qubit working solution (see Step 23.1)
Open and label additional tubes needed from Qiagen DNeasy Blood & Tissue Kit:
- Packaged spin columns: # of samples + extraction blank
- Additional collection tubes: (# of samples + extraction blank) * 2
Wipe down 1000 µl pipette, 200 µl pipette, 100 µl pipette, 10 µl pipette with RNase Away and UV for 15 minutes on each side
Place 50 mL tube of molecular-grade ethanol in freezer or on ice to chill for later use
Heat aliquot (volume calculation below) of AE buffer (from Qiagen DNeasy Blood & Tissue Kit) on heating block at 70°C
Volume: (# of samples including blank * 100 µl) * 1.1
Make cryo-labels for storage tubes (2 of 3 1.5/2 mL LoBind tubes already sterilized)
Assemble other reagents & materials needed:
- Sterile 3 mL syringes (one per sample)
- AL Buffer (from Qiagen DNeasy Blood & Tissue Kit)
- Buffer AW1 (from Qiagen DNeasy Blood & Tissue Kit)
- Buffer AW2 (from Qiagen DNeasy Blood & Tissue Kit)
Remove Sterivex filters from incubator
Remove liquid from each filter:
Handshake vigorously for several seconds
Remove caps from filter
Using a sterile 3 mL syringe attached to the inlet end of the Sterivex, remove all liquid from filter, record the volume, and transfer into a 5 mL LoBind tube
Create an extraction blank by adding 1000 uL nuclease free water subbed in for extracted liquid to a 5 mL LoBind tube
For each sample and extraction blank, add AL buffer and 0°C ethanol to the extracted liquid in a 1:1:1 ratio and vortex vigorously for 10 seconds
For each sample and extraction blank, filter the mixture through a spin column:
Pipet the mixture (650 µl at a time) into a DNeasy Mini Spin column placed in a collection tube
Spin in micro-centrifuge for 1 minute at 6000 x g (8000 rpm)
Discard flow throw, and dab the rim of the spin column dry on a Kimwipe
Repeat sub-steps of 22 until all sample is filtered through spin column
For each sample and extraction blank follow the remaining Qiagen DNeasy Tissue and Blood Kit steps:
Place the spin column in a new collection tube, add 500 µl Buffer AW1
Centrifuge for 1 minute at 6000 x g (8000 rpm). Discard flow through and collection tube.
Place in new collection tube and add 500 µl Buffer AW2
Centrifuge for 3 min at 20,000 x g (14000 rpm) to dry the membrane
Discard flow through and dab rim of spin column on a clean Kimwipe
Place the spin column back in collection tube and centrifuge for 1 min at 20,000 x g (14000 rpm)
Transfer spin column to new 1.5/2 mL LoBind tube with cap left open
Place samples in 70°C heat block
Add 100 µl Buffer AE (4 samples at a time) directly over membrane
Immediately transfer tubes to room temperature
Incubate at room temperature for 10 minutes
Centrifuge for 1 min at 6000 x g (8000 rpm)
Re-elute DNA from DNA LoBind tube (apply eluate back on spin column while tubes are in heat block)
Incubate at room temperature for 10 minutes
Centrifuge for 1 min at 6000 x g (8000 rpm)
Discard the spin column
Note
Sample tubes should now contain a final volume of 100 µl of DNA extract
Aliquot 1 µl of each sample into labeled Qubit tubes
Transfer ~50 µl of remaining DNA extract to 2 pre-marked DNA LoBind tubes with lids intact:
- 1 archive tube to store at -80°C
- 1 working tube to store at -15°C
Note
Aliquot to archive tube first, so that archive volumes are consistently 50 µl. Working tube volumes may be slightly less than 50 µl due to Qubit aliquot, etc.
Use Qubit to measure DNA concentrations in all samples:
Make Qubit working solution:
- Reagent: ((# of samples + extraction blank + 2) * 1 )*1.1
- Buffer: ((# of samples + extraction blank + 2) * 199)*1.1
Vortex Qubit working solution
Add 199 µl of working solution to Qubit tubes containing sample DNA
Add 190 µl of working solution to Qubit tubes for 2 standards
Add 10 µl of respective standards to Qubit tubes for 2 standards
Mix all tubes gently by vortexing, being sure not to introduce bubbles
Allow tubes to incubate at room temperature for 2 minutes
Read samples with Qubit fluorometer and record DNA concentrations
Protocol references
Qiagen DNeasy Blood & Tissue Handbook, July 2020, Qiagen. Available online and uploaded as a high-level attachment to this protocol.
Spens, J., Evans, A.R., Halfmaerten, D., Knudsen, S.W., Sengupta, M.E., Mak, S.S.T., Sigsgaard, E.E., Hellstrom, M., 2017. Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. METHODS IN ECOLOGY AND EVOLUTION 8, 635–645. https://doi.org/10.1111/2041-210X.12683