Available via license: CC BY 4.0
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AUG 01, 2023
DNA Extraction Protocol from Sterivex Filters
Meghan M. Shea ,
Alexandria B
Boehm
Stanford University
Meghan M. Shea
Stanford University
DOI:
dx.doi.org/10.17504/protocol
s.io.ewov1qyyygr2/v1
Protocol Citation: Meghan
M. Shea, Alexandria B Boehm
2023. DNA Extraction
Protocol from Sterivex Filters.
protocols.io
https://dx.doi.org/10.17504/p
rotocols.io.ewov1qyyygr2/v1
MANUSCRIPT CITATION:
TBD
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
1 1
1
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
NAME
Coastal Environmental DNA Sampling & Gravity Filtration
Protocol
CREATED BY
Meghan M. Shea
PREVIEW
ATTACHMENTS
HB-2061-
003_HB_DNY_Blood_Tiss
ue_0720_WW.pdf
IMAGE ATTRIBUTION
Meghan M. Shea
MATERIALS
General Laboratory Equipment:General Laboratory Equipment:
Equipment
Specific Model Used
protocols.io |
https://dx.doi.org/10.17504/protocols.io.ewov1qyyygr2/v1 1
Created: Jun 16, 2023
Last Modified: Aug 01,
2023
PROTOCOL integer ID:
83545
Keywords: environmental
DNA, DNA extraction,
Sterivex, Qiagen DNeasy
Blood and Tissue Kit
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/vw
r-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/pola
rsafe-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
Equipment
Specific Model Used
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Note
Additional Materials:Additional Materials:
Material
Amount Needed
Source
Link
Approx. Cost
Qubit dsDNA
Broad Range
assay kit
1
reaction/sampl
e + additional
for standards
Fisher
Scientific
(Q32850)
https://www.fis
hersci.com/sho
p/products/qubi
t-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.fis
hersci.com/sho
p/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.fis
hersci.com/sho
p/products/wat
er-dna-grade-
dnase-protease-
free-fisher-
bioreagents/BP
24701
$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.us
ascientific.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.fis
hersci.com/sho
p/products/eth
anol-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.us
ascientific.com/
eppendorf-tube-
5-0-ml-dna-
lobind/p/4011-
8310
$68.5/200
tubes
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.
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Qiagen
DNeasy Blood
& Tissue Kit
1
reaction/sampl
e
Qiagen
(69506)
https://www.qi
agen.com/us/pr
oducts/discover
y-and-
translational-
research/dna-
rna-
purification/dna
-
purification/gen
omic-
dna/dneasy-
blood-and-
tissue-kit?
catno=69506
$692.3/250
preparations
Qubit tubes
1/sample +
additional for
standards
Unknown
NA
Unknown
Material
Amount Needed
Source
Link
Approx. Cost
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.
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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
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.
Caution: DO NOT add bleach or acidic solutions directly to the
sample preparation waste.
1Clean bench area with bleach, ethanol, and RNase away
2Turn on incubator and set to 56°C
3Wipe down 1000 µL and 200 µL pipettes with RNase Away and UV for 15 minutes on each side
Day 1 - DNA Lysing
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4Assemble 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)
5Remove 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.
6For each filter:
6.1 Remove Sterivex from Whirl-Pak bag and remove cap from inlet end of Sterivex filter
6.2 Slowly pipette 80 µL of Proteinase K directly on top of the filter through the inlet, avoiding
backsplash by expelling slowly
6.3 Slowly pipette 720 µL of ATL buffer directly on top of the filter through the inlet, again
avoiding backsplash
6.4 Secure Sterivex with same luer cap
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8Incubate 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
9Clean bench area (including vortex), centrifuge area, and centrifuge with bleach, ethanol, and
RNase away.
10 Soak all tube racks in a 10% bleach solution, followed by 3 rinses with DI water. Let dry and UV
for 15 minutes
11 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)
12 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
13 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
14 Place 50 mL tube of molecular-grade ethanol in freezer or on ice to chill for later use
Day 2 - DNA Extraction
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15 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
16 Make cryo-labels for storage tubes (2 of 3 1.5/2 mL LoBind tubes already sterilized)
17 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)
18 Remove Sterivex filters from incubator
19 Remove liquid from each filter:
19.1 Handshake vigorously for several seconds
19.2 Remove caps from filter
19.3 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
20 Create an extraction blank by adding 1000 uL nuclease free water subbed in for extracted liquid
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to a 5 mL LoBind tube
21 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
22 For each sample and extraction blank, filter the mixture through a spin column:
22.1 Pipet the mixture (650 µl at a time) into a DNeasy Mini Spin column placed in a collection tube
22.2 Spin in micro-centrifuge for 1 minute at 6000 x g (8000 rpm)
22.3 Discard flow throw, and dab the rim of the spin column dry on a Kimwipe
22.4 Repeat sub-steps of 22 until all sample is filtered through spin column
23 For each sample and extraction blank follow the remaining Qiagen DNeasy Tissue and Blood Kit
steps:
23.1 Place the spin column in a new collection tube, add 500 µl Buffer AW1
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23.2 Centrifuge for 1 minute at 6000 x g (8000 rpm). Discard flow through and collection tube.
23.3 Place in new collection tube and add 500 µl Buffer AW2
23.4 Centrifuge for 3 min at 20,000 x g (14000 rpm) to dry the membrane
23.5 Discard flow through and dab rim of spin column on a clean Kimwipe
23.6 Place the spin column back in collection tube and centrifuge for 1 min at 20,000 x g (14000
rpm)
23.7 Transfer spin column to new 1.5/2 mL LoBind tube with cap left open
23.8 Place samples in 70°C heat block
23.9 Add 100 µl Buffer AE (4 samples at a time) directly over membrane
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23.10 Immediately transfer tubes to room temperature
23.11 Incubate at room temperature for 10 minutes
23.12 Centrifuge for 1 min at 6000 x g (8000 rpm)
23.13 Re-elute DNA from DNA LoBind tube (apply eluate back on spin column while tubes are in
heat block)
23.14 Incubate at room temperature for 10 minutes
23.15 Centrifuge for 1 min at 6000 x g (8000 rpm)
23.16 Discard the spin column
Note
Sample tubes should now contain a final volume of 100 µl of DNA extract
24 Aliquot 1 µl of each sample into labeled Qubit tubes
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25 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.
26 Use Qubit to measure DNA concentrations in all samples:
26.1 Make Qubit working solution:
Reagent: ((# of samples + extraction blank + 2) * 1 )*1.1
Buffer: ((# of samples + extraction blank + 2) * 199)*1.1
26.2 Vortex Qubit working solution
26.3 Add 199 µl of working solution to Qubit tubes containing sample DNA
26.4 Add 190 µl of working solution to Qubit tubes for 2 standards
26.5 Add 10 µl of respective standards to Qubit tubes for 2 standards
26.6 Mix all tubes gently by vortexing, being sure not to introduce bubbles
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