TITLE: DNA Extraction Protocol from Sterivex Filters
AUTHORS: Meghan M. Shea, Alexandria B Boehm

[DESCRIPTION]
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: 
 


[STEPS]
SECTION: Day 1 - DNA Lysing
1. Clean bench area with bleach, ethanol, and RNase away


SECTION: Day 1 - DNA Lysing
2. Turn on incubator and set to 56°C
SECTION: Day 1 - DNA Lysing
3. Wipe down 1000 µL and 200 µL pipettes with RNase Away and UV for 15 minutes on each side
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5. Remove sterivex filters from -15°C freezer
 

SECTION: Day 1 - DNA Lysing
6. For each filter:
SECTION: Day 1 - DNA Lysing
6.1. Remove Sterivex from Whirl-Pak bag and remove cap from inlet end of Sterivex filter 
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6.3. Slowly pipette 720 µL of ATL buffer directly on top of the filter through the inlet, again avoiding backsplash
SECTION: Day 1 - DNA Lysing
6.4. Secure Sterivex with same luer cap
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6.5. Handshake vigorously for several seconds
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7. Place all filters onto roller shaker in incubator
 
SECTION: Day 1 - DNA Lysing
8. 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
SECTION: Day 1 - DNA Lysing
6.2. Slowly pipette 80 µL of Proteinase K directly on top of the filter through the inlet, avoiding backsplash by expelling slowly
SECTION: Day 2 - DNA Extraction
10. Soak all tube racks in a 10% bleach solution, followed by 3 rinses with DI water. Let dry and UV for 15 minutes
SECTION: Day 2 - DNA Extraction
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)

SECTION: Day 2 - DNA Extraction
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
SECTION: Day 2 - DNA Extraction
14. Place 50 mL tube of molecular-grade ethanol in freezer or on ice to chill for later use
SECTION: Day 2 - DNA Extraction
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
SECTION: Day 2 - DNA Extraction
16. Make cryo-labels for storage tubes (2 of 3 1.5/2 mL LoBind tubes already sterilized)
SECTION: Day 2 - DNA Extraction
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
SECTION: Day 2 - DNA Extraction
18. Remove Sterivex filters from incubator
SECTION: Day 2 - DNA Extraction
19. Remove liquid from each filter:
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19.1. Handshake vigorously for several seconds
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19.2. Remove caps from filter
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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
SECTION: Day 2 - DNA Extraction
22.1. Pipet the mixture (650 µl at a time) into a DNeasy Mini Spin column placed in a collection tube
SECTION: Day 2 - DNA Extraction
22.2. Spin in micro-centrifuge for 1 minute at 6000 x g (8000 rpm)
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22.3. Discard flow throw, and dab the rim of the spin column dry on a Kimwipe
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22.4. Repeat sub-steps of 22 until all sample is filtered through spin column
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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.
SECTION: Day 2 - DNA Extraction
23.3. Place in new collection tube and add 500 µl Buffer AW2
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23.4. Centrifuge for 3 min at 20,000 x g (14000 rpm) to dry the membrane
SECTION: Day 2 - DNA Extraction
23.5. Discard flow through and dab rim of spin column on a clean Kimwipe
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23.6. Place the spin column back in collection tube and centrifuge for 1 min at 20,000 x g (14000 rpm)
SECTION: Day 2 - DNA Extraction
23.7. Transfer spin column to new 1.5/2 mL LoBind tube with cap left open
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23.8. Place samples in 70°C heat block
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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
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23.11. Incubate at room temperature for 10 minutes
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23.12. Centrifuge for 1 min at 6000 x g (8000 rpm)
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23.13. Re-elute DNA from DNA LoBind tube (apply eluate back on spin column while tubes are in heat block)
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23.14. Incubate at room temperature for 10 minutes
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23.15. Centrifuge for 1 min at 6000 x g (8000 rpm)
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23.16. Discard the spin column

 

SECTION: Day 2 - DNA Extraction
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
 

SECTION: Day 2 - DNA Extraction
26. Use Qubit to measure DNA concentrations in all samples:
SECTION: Day 2 - DNA Extraction
26.1. Make Qubit working solution:
Reagent: ((# of samples + extraction blank + 2) * 1 )*1.1
Buffer: ((# of samples + extraction blank + 2) * 199)*1.1
SECTION: Day 2 - DNA Extraction
26.2. Vortex Qubit working solution
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26.3. Add 199 µl of working solution to Qubit tubes containing sample DNA
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26.4. Add 190 µl of working solution to Qubit tubes for 2 standards
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26.5. Add 10 µl of respective standards to Qubit tubes for 2 standards
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26.6. Mix all tubes gently by vortexing, being sure not to introduce bubbles
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26.7. Allow tubes to incubate at room temperature for 2 minutes
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26.8. Read samples with Qubit fluorometer and record DNA concentrations
SECTION: Day 2 - DNA Extraction
23. For each sample and extraction blank follow the remaining Qiagen DNeasy Tissue and Blood Kit steps:
SECTION: Day 1 - DNA Lysing
4. 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)
SECTION: Day 2 - DNA Extraction
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)
SECTION: Day 2 - DNA Extraction
9. Clean bench area (including vortex), centrifuge area, and centrifuge with bleach, ethanol, and RNase away. 
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20. Create an extraction blank by adding 1000 uL nuclease free water subbed in for extracted liquid to a 5 mL LoBind tube
SECTION: Day 2 - DNA Extraction
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
SECTION: Day 2 - DNA Extraction
22. For each sample and extraction blank, filter the mixture through a spin column: