TITLE: DNA extraction from environmental biofilm using the NucleoSpin® Soil kit (MACHEREY-NAGEL) 
AUTHORS: Marine Vautier, Valentin Vasselon, Cecile Chardon, Frédéric Rimet, Agnès Bouchez, Isabelle Domaizon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol is part of the DNA workflow applied in the Eco-AlpsWater Project.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The methodological step described here is the extraction of DNA, this is a critical step for obtaining relevant results since molecular inventories might be influenced by the DNA extraction method used.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The choice of the methodology for biofilms DNA extraction is based on previous studies and in particular on the work done by Vasselon et al. (2017) where the authors have tested 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA (from pure diatom cultures and biofilms samples from streams and lakes).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">For the Eco-AlpsWater project, after being sampled in lakes or rivers, biofilms are stored in 50 mL tubes in ethanol at 4°C, and for a maximum of 3 months before DNA extraction (the extraction should preferably be done in the month following the sampling).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The DNA extraction protocol presented below has been used in several recent studies (e.g. Vasselon et al 2017ab, 2018) focussed on the application of diatoms metabarcoding; this extraction is based on a protocol adapted from the NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications for biofilm DNA extraction.</div></div></div>

[STEPS]
?. [Prepare the sample]
Shake the 50mL falcon tube containing the biofilm / ethanol mixture by inverting the tube in order to obtain a homogeneous solutionTake 2 mL of this homogeneized solution by pipetting twice 1 mL into a 2 mL sterile microcentrifuge tube and close the capNote : between each pipetting, aspirate/discharge to resuspend the biofilm particles that may have sedimented at the bottom of the tubeCentrifuge   Discard the supernantantAdd   of buffer SL1 to the biofilm pelletNote : check SL1 buffer - dissolve any precipitate by incubating the buffer at 30-40°C for 10 min and shaking the bottle every 2 min Resuspend the biofilm pelletTransfer the biofilm/SL1 mixture into the NucleoSpin®  Bead Tube Type ANote : between each pipetting, aspirate/discharge to resuspend the biofilm particles that may have sedimented at the bottom of the tubeCentrifuge Discard the supernantantAdd   of buffer SL1 to the biofilm pelletNote : check SL1 buffer - dissolve any precipitate by incubating the buffer at 30-40°C for 10 min and shaking the bottle every 2 min Resuspend the biofilm pelletTransfer the biofilm/SL1 mixture into the NucleoSpin®  Bead Tube Type AAdd  of buffer SL1 to the biofilm pelletNote : check SL1 buffer - dissolve any precipitate by incubating the buffer at 30-40°C for 10 min and shaking the bottle every 2 min Resuspend the biofilm pelletTransfer the biofilm/SL1 mixture into the NucleoSpin®  Bead Tube Type A
Centrifuge: 18000 34, 30 min, +4 10
700 µl
?. [Prepare the sample]
?. [Adjust lysis conditions]
Add of Enhancer SX
150 µl
?. [Sample lysis]
Note : using a special adapter or alternatively tape to fix the tubeVortex the samples at full speed at  (18 - 25°C) for
0 Room temperature
?. [Precipitate contaminants]
Centrifuge Add  of buffer SL3 and vortex for Incubate at   for Centrifuge
Centrifuge: 11000 34, 2 min
150 µl
[in a fridge]
Centrifuge: 11000 34, 1 min
?. [Filter lysate]
Place a NucleoSpin®  Inhibitor Removal Column (red ring) in a Collection Tube (2mL, lid)Load up to   of clear supernatant (obtained at the step 4) onto the filterCentrifuge   Repeat the load and the centrifuge step as many time as there is still some supernatant from step 4 to be filteredAfter each centrifugation, collect the filtered liquid in a clean tube : 1 single tube for all the filtrationDiscard the NucleoSpin®  Inhibitor Removal Column Load up to  of clear supernatant (obtained at the step 4) onto the filterCentrifuge Repeat the load and the centrifuge step as many time as there is still some supernatant from step 4 to be filteredAfter each centrifugation, collect the filtered liquid in a clean tube : 1 single tube for all the filtrationDiscard the NucleoSpin®  Inhibitor Removal Column After each centrifugation, collect the filtered liquid in a clean tube : 1 single tube for all the filtrationNote : if a pellet is visible after the centrifugation, transfer the clear supernantant to a new collection tube (not provided in the kit) to get ride of this pellet, and continue with the clear supernatant
650 µl
Centrifuge: 11000 34, 1 min
?. [Adjust binding conditions]
Add  of buffer SB Vortex for  , make a brief centrifugation
250 µl
?. [Bind DNA]
Place a NucleoSpin®  Soil Column (green ring) in a collection Tube ( 2mL)Load   of sample onto the columnCentrifuge   Discard the flow through and place the column back into the collection tubeLoad the remaining sample onto the columnCentrifuge   Discard the flow through and place the column back into the collection tubeLoad  of sample onto the columnCentrifuge Discard the flow through and place the column back into the collection tubeLoad the remaining sample onto the columnCentrifuge   Discard the flow through and place the column back into the collection tubeCentrifuge
550 µl
Centrifuge: 11000 34, 1 min
Centrifuge: 11000 34, 1 min
?. [Wash and dry silica membrane]
Note : the same collection tube is used throughout the entire washing procedure to reduce plastic waste
?. [Wash and dry silica membrane]
1st wash : Add  of buffer SB to the NucleoSpin®  Soil ColumnCentrifuge
500 µl
Centrifuge: 11000 34, 30 s
?. [Wash and dry silica membrane]
2nd wash : Add  of buffer SW1 to the NucleoSpin®  Soil ColumnCentrifuge
550 µl
Centrifuge: 11000 34, 30 s
?. [Wash and dry silica membrane]
3rd wash : Add  of buffer SW2 to the NucleoSpin®  Soil ColumnNote : verify that ethanol was added to buffer SW2 during the first utilisationCentrifuge
650 µl
Centrifuge: 11000 34, 30 s
?. [Wash and dry silica membrane]
4th wash : Add  of buffer SW2 to the NucleoSpin®  Soil ColumnCentrifuge
650 µl
Centrifuge: 11000 34, 30 s
?. [Dry silica membrane]
Centrifuge Note : if for any reason, the liquid in the collection tube has touched the  NucleoSpin®  Soil Column after drying step, discard flow through and centrifuge again
Centrifuge: 11000 34, 2 min
?. [Elute DNA]
Place the NucleoSpin®  Soil Column into a new microcentrifuge tube (not provided in the kit)Add    of buffer SE previously heated   to the columnDo not close the lid and incubate at   for   Close the lid and centrifuge   Discard the NucleoSpin®  Soil Column and keep the tube cointaining the DNAWe recommend storing DNA frozen at -20°C until preparation of DNA library for HTS (or at -40°C to -80°C for longer storage)Add   of buffer SE previously heated  to the columnDo not close the lid and incubate at  for Close the lid and centrifuge Discard the NucleoSpin®  Soil Column and keep the tube cointaining the DNAWe recommend storing DNA frozen at -20°C until preparation of DNA library for HTS (or at -40°C to -80°C for longer storage)
30 µl
50 °C
0 Room temperature
Centrifuge: 11000 34, 30 s