TITLE: Fish eDNA: DNA extraction from water samples filtered through Sterivex filter unit and preserved in buffer 
AUTHORS: Marine  Vautier, Cécile Chardon, Isabelle Domaizon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>The  objective of this protocol is DNA extraction from water samples filtered through 0.45 µm Sterivex™ filter units and preserved in 2  mL of buffer (</span><a href="https://dx.doi.org/10.17504/protocols.io.br5rm856" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.br5rm856</span></a><span>). </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Extraction is performed using NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications adapted to DNA extraction from Sterivex™ filter units.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol is used upstream to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target fish eDNA.</div></div></div>

[STEPS]
?. [Prepare the sample]
Note : If the samples have been stored at RT or at 4°C, proceed directly to step 2. If the samples have been frozen, proceed to step 1.Defrost the sample at   (about  )Note : during this step heat the incubator at
4 °C
37 °C
?. [Sample Lysis]
Fix the Sterivex cartriges on the vortex and vortex for   to remove particles from the filterRemove the Inlet Cap (Figure 1) and with a pipette tip, add   of lysozyme solution into the Sterivex already filled with 2mL of buffer. Insert pipette completely into the inlet to see the pipette tip inside the unit just above the membrane. Re-cap the Sterivex.Rotate for  at  (during this step, place the SDS solution in the incubator for the next step)Remove the Inlet Cap and and with a pipette tip, add  of Proteinase K solution and  of SDS 20%. Re-cap the Sterivex.Note : during this step heat the incubator at Incubate for  at   with agitation (or shake manually every 15 min). Remove the Inlet Cap and fix a sterile syringe to the Sterivex. Aspirate the liquid contained in the Sterivex with the syringe by moving back and forth to collect all the liquidTransfer the lysate into two 2 mL tubes (about 1.1 mL per tube).Aspirate the liquid contained in the Sterivex with the syringe by moving back and forth to collect all the liquidTransfer the lysate into two 2 mL tubes (about 1.1 mL per tube).Note : after this step, place the Buffer SE in the incubator heated at    to anticipate the elution step
40 µl
37 °C
50 µl
100 µl
55 °C
55 °C
55 °C
?. [Precipitate contaminants]
In both 2mL tubes,  add   of buffer SL3.Vortex for Incubate for   at Centrifuge at
200 µl
[in a fridge]
Centrifuge: 11000 34, 2 min
?. [Filter lysate]
Place a NucleoSpin® Inhibitor Removal Column (red ring) in a collection Tube (2 mL, lid).Load up to    of clear supernatant (obtained at the step 3) onto the filter.Centrifuge at   Remove the column and close the tube containing the flow through.Repeat these steps as many time as there is still some supernatant from step 3 to be filtered. For each centrifugation, collect the supernatant in a clean tube (about 4 tubes are required, but only 1 column for the filtration of all lysate from step 3).Discard the NucleoSpin® Inhibitor Removal Column.Load up to   of clear supernatant (obtained at the step 3) onto the filter.Centrifuge at Remove the column and close the tube containing the flow through.Repeat these steps as many time as there is still some supernatant from step 3 to be filtered. For each centrifugation, collect the supernatant in a clean tube (about 4 tubes are required, but only 1 column for the filtration of all lysate from step 3).Discard the NucleoSpin® Inhibitor Removal Column.Repeat these steps as many time as there is still some supernatant from step 3 to be filtered. For each centrifugation, collect the supernatant in a clean tube (about 4 tubes are required, but only 1 column for the filtration of all lysate from step 3).Discard the NucleoSpin® Inhibitor Removal Column.Note: If a pellet is visible after the centrifugation, transfer the clear supernatant to a new collection tube (not provided in the kit) to get rid of this pellet, and continue with the clear supernatant.
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650 µl
Centrifuge: 11000 34, 1 min
?. [Adjust binding conditions]
Add  of Buffer SB per 2mL tube.Note : 0.38V of buffer SB for 1V of flow through from step 4.Close the lid.Vortex for  and centrifuge briefly.
250 µl
?. [Bind DNA]
Place a NucleoSpin® Soil Column (green ring) in a Collection tube (2 mL).Load    of sample from step 5 onto the column.Centrifuge  at   Discard the flow through and place the column back into the collection tube.Repeat the three previous steps with all the tubes from step 5 and using the same column.Discard the flow through and place the column back into the collection tube.Load   of sample from step 5 onto the column.Centrifuge  at Discard the flow through and place the column back into the collection tube.Repeat the three previous steps with all the tubes from step 5 and using the same column.Discard the flow through and place the column back into the collection tube.Repeat the three previous steps with all the tubes from step 5 and using the same column.Discard the flow through and place the column back into the collection tube.
650 µl
Centrifuge: 11000 34, 1 min
?. [Wash silica membrane]
Note : the same collection tube is used throughout the entire washing procedure to reduce plastic waste.
?. [Wash silica membrane]
1st wash :  Add  of Buffer SB to the NucleoSpin® Soil Column.Centrifuge at
500 µl
Centrifuge: 11000 34, 30 s
?. [Wash silica membrane]
2nd Wash :Add  of Buffer SW1 to the NucleoSpin® Soil Column.Centrifuge at 
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550 µl
Centrifuge: 11000 34, 30 s
?. [Wash silica membrane]
3rd Wash :Add  of Buffer SW2 to the NucleoSpin® Soil Column.Vortex  and centrifuge at   .
650 µl
Centrifuge: 11000 34, 30 s
?. [Wash silica membrane]
4th Wash : Add   of Buffer SW2 to the NucleoSpin® Soil Column.Vortex  and centrifuge at  .
650 µl
Centrifuge: 11000 34, 30 s
?. [Dry silica membrane]
Centrifuge at   .Note: if for any reason, the liquid in the collection tube touched the NucleoSpin® Soil Column after the drying step, discard flow through and centrifuge again.
Centrifuge: 11000 34, 2 min
?. [Elute DNA]
Place the NucleoSpin® Soil Column into a new 1.5mL microcentrifuge tube.Add    of Buffer SE previously heated to 55°C into the column.Note: a larger volume of SE buffer can be used but the DNA concentration will be lowerDo not close the lid and incubate for   at   (18-25°C).Close the lid and centrifuge at  Throw the column and keep the tube containing the DNA.Note: A second elution can be performed with the same column to collect more DNA (the concentration will be lower for the second elution)We recommend storing DNA frozen at -20°C until the use of the DNA sample (or at -40°C to -80°C for longer storage).Add   of Buffer SE previously heated to 55°C into the column.Note: a larger volume of SE buffer can be used but the DNA concentration will be lowerDo not close the lid and incubate for  at  (18-25°C).Close the lid and centrifuge at Throw the column and keep the tube containing the DNA.Note: A second elution can be performed with the same column to collect more DNA (the concentration will be lower for the second elution)We recommend storing DNA frozen at -20°C until the use of the DNA sample (or at -40°C to -80°C for longer storage).Note: A second elution can be performed with the same column to collect more DNA (the concentration will be lower for the second elution)
30 µl
0 Room temperature
Centrifuge: 11000 34, 30 s