TITLE: Sample collection and eDNA extraction from Sterivex filter units
AUTHORS: Oscar Chiang, Pedro Inostroza

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following workflow covers several steps in the DNA analysis of environmental samples, from the water collection to the analysis back in the lab. The samples can be taken from several water systems (i.e. sea, lakes, rivers, streams) and collected in triplicate (1 L) in </span><a href="https://www.merckmillipore.com/SE/en/product/Sterivex-Filter-Units,MM_NF-C3235?ReferrerURL=https%3A%2F%2Fduckduckgo.com%2F&bd=1#overview" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Sterivex sterile filter units</span></a><span> (Merck, cat. no. SVGP01050). The DNA extraction protocol modifies the </span><a href="https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powerwater-sterivex-kit/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dneasy PowerWater Sterivex kit</span></a><span> (Qiagen, cat. no. 14600-50-nf).</span></div><br/></div>

[STEPS]
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Place the Sterivex vertically (with the inlet cap upward) and load of ST1B buffer. ST1B is stored at . Be careful putting the pipette tip through the inlet (orange cap). Dispense the buffer slowly; a fraction of the volume can be lost.
900 µl
4 °C
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Allow mixing in a vortex with a horizontal adapter (Qiagen, Cat. No. 13000-V1-5) for 00:05:00 at the minimum speed. Set the filters with the inlet facing out and check for potential leakage from the outlet sealed with Parafilm.
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Rotate the Sterivex in 90 degrees and vortex for  at the minimum speed. Repeat the previous step 2 more times (4 times x  each filter)
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Add  of solution MBL. Dispense slowly; a fraction of the volume can be lost. Before use, heat the MBL solution at 65°C for as is suggested by the manufacturer.
900 µl
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Incubate the filter units vertically with the inlet upward at for in an oven. Before and after the incubation, check for any leakage in the Parafilm; replace it if needed.
90 °C
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Let the filters cool down at room temperature for , and re-tight the caps and check the Parafilm. Then, vortex at maximum speed for . While mixing, check that the filters stay in place. If not, lower the speed.
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Transfer the lysate from the filter unit to a  syringe; push 1 mL of air into the filter while it is vertical, and then release the plunger. Continue pulling back until the lysate is recovered in the syringe.
3 mL
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Pour the lysate into a Powerbeat tube and vortex horizontally for  at maximum speed
5 mL
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Centrifuge the tube at for
Centrifuge: 4000 34
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Carefully transfer the lysate to a clean  collection tube. Then, add  of RNAse (final concentration ). Incubate the samples at  x  in a heating block or a water bath.
2.2 mL
1 µl
37 °C
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Add  IRS solution to the tube and vortex. Then incubate at  for .
300 µl
4 °C
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Centrifuge the tube at for
Centrifuge: 13000 34
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Transfer the supernatant to a  collection tube, avoiding the pellet, and add  of MR solution and vortex. Heat the MR solution at for  before using it.
5 mL
3 mL
65 °C
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Load the supernatant () into a tube extender and MB spin column. Filtrate at low pressure. The column is attached to the VacValve vacuum system, as shown below.
4 mL
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Once the lysate passes through, the column extender is carefully removed. Next, wash the spin column with of ethanol. Keep the columns open.
800 µl
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Repeat step 21, and wash the spin column adding of ethanol. Allow the membrane to dry as before.
800 µl
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Place the spin column in a clean collection tube and dry it out by centrifugation at  for
Centrifuge: 13000 34
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Transfer the spin column to a new clean tube, and add of solution EB (or DNA-free gradewater). Centrifuge at  for .
50 µl
Centrifuge: 13000 34
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The DNA purity is checked with a Nanodrop.
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A Sterivex filter unit (0.22 µm pore size) is attached to the suction hose by a stainless steel male Luer-lock ring hose barb (1/4'). We recommend using a hose clip (9-10 mm; Buerkle cat. no. BURK8678-000) to firmly tight the Luer-lock ring to the hose. Each filter can process up to 2L of water depending on the amount of the suspended material.
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Voila! The DNA is in the collection tube, ready for further processing.
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The DNA concentration is quantified by Qubit.
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Once the water sample is filtered, remove the remaining liquid from the Sterivex unit using a 5 mL sterilized syringe or similar by pushing air.
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A portable peristaltic pump (Vampire sampler; Buerkle) is used for sampling at an approximate 30-50 mL/min flow rate. The tubing inserted in the unit's head has a suction hose (Marprene; 4.8 mm inner diameter), connected to a flexible silicone hose. A suitable adapter connects both hoses.
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Cap both ends of the Sterivex. If the units have a male tip outlet, Parafilm can be used to cap them. Be aware of potential leakage, and make sure the Sterivex is dried and well sealed.
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Now that the samples are ready and sealed, do not forget to identify them accordingly. A good rule of thumb is to write down ID, date, replicate number, filtered volume and station on the filter. Do the same on the sample bags, especially if there are more filters per site or replicate. The samples are store at  degrees.
-20 °C
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Now, wash the spin column with of PW solution. Keep the columns open. Allow the membrane to dry by keeping the vacuum pump running for 1 min.
800 µl
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A VacValve vacuum system and a vacuum pump are used for the following steps.