TITLE: Extracellular DNA extraction from sediment using phosphate buffer and NucleoSpin® Soil kit (MACHEREY NAGEL)
AUTHORS: Cecile Chardon, Louis Jacas, Isabelle Domaizon

[DESCRIPTION]
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[STEPS]
?. [Prepare the sample]
Annotate 2mL tubes, one 2mL tube per sampleWeigh annotated tubes, use the weigh scale (0.0001g of precision)         → On your notebook, note the name of tubes and their weightsWith a sterilized spatula, homogenize a sediment and check that it is completely thawed (if not, wait for total defrost)Transfer ≈ 0.5g of sediment in the associated tube, 2 methods :         -    if the sediment is "compact", use a sterilized spatula to transfer the sediment        -    if the sediment countains a lot of water, use a 1000µL tip cuted with clean chisel and transfer sediment by pipetting few times.          → On your notebook, note the used method and your comments about the sediment particularityWeigh the tube containing sediment        → On your notebook, note the weightOn the storage tube, make a cross to show that this tube countains tawed sediment thaw once : this is highlyrecommended to do not subject sediment to freeze-thaw cycles in particular if the aim is to perform downstream analyses on sed-DNA extracts.Keep this sediment at -40°C or  -80°C for long term storage.        → On your notebook, note where the sediments are storedRepeat this process for all the sediments that will be extracted during this sessionAdd one volume of saturated phosphate buffer (0.12 M Na2HPO4; pH ≈ 8; stored less than one week at +4°C) for one volume of sedimente.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate bufferClose the cap and you can add parafilm to secure the closure         → On your notebook, note the name of tubes and their weightsWith a sterilized spatula, homogenize a sediment and check that it is completely thawed (if not, wait for total defrost)Transfer ≈ 0.5g of sediment in the associated tube, 2 methods :         -    if the sediment is "compact", use a sterilized spatula to transfer the sediment        -    if the sediment countains a lot of water, use a 1000µL tip cuted with clean chisel and transfer sediment by pipetting few times.          → On your notebook, note the used method and your comments about the sediment particularityWeigh the tube containing sediment        → On your notebook, note the weightOn the storage tube, make a cross to show that this tube countains tawed sediment thaw once : this is highlyrecommended to do not subject sediment to freeze-thaw cycles in particular if the aim is to perform downstream analyses on sed-DNA extracts.Keep this sediment at -40°C or  -80°C for long term storage.        → On your notebook, note where the sediments are storedRepeat this process for all the sediments that will be extracted during this sessionAdd one volume of saturated phosphate buffer (0.12 M Na2HPO4; pH ≈ 8; stored less than one week at +4°C) for one volume of sedimente.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate bufferClose the cap and you can add parafilm to secure the closure        -    if the sediment is "compact", use a sterilized spatula to transfer the sediment        -    if the sediment countains a lot of water, use a 1000µL tip cuted with clean chisel and transfer sediment by pipetting few times.          → On your notebook, note the used method and your comments about the sediment particularityWeigh the tube containing sediment        → On your notebook, note the weightOn the storage tube, make a cross to show that this tube countains tawed sediment thaw once : this is highlyrecommended to do not subject sediment to freeze-thaw cycles in particular if the aim is to perform downstream analyses on sed-DNA extracts.Keep this sediment at -40°C or  -80°C for long term storage.        → On your notebook, note where the sediments are storedRepeat this process for all the sediments that will be extracted during this sessionAdd one volume of saturated phosphate buffer (0.12 M Na2HPO4; pH ≈ 8; stored less than one week at +4°C) for one volume of sedimente.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate bufferClose the cap and you can add parafilm to secure the closure        → On your notebook, note the weightOn the storage tube, make a cross to show that this tube countains tawed sediment thaw once : this is highlyrecommended to do not subject sediment to freeze-thaw cycles in particular if the aim is to perform downstream analyses on sed-DNA extracts.Keep this sediment at -40°C or  -80°C for long term storage.        → On your notebook, note where the sediments are storedRepeat this process for all the sediments that will be extracted during this sessionAdd one volume of saturated phosphate buffer (0.12 M Na2HPO4; pH ≈ 8; stored less than one week at +4°C) for one volume of sedimente.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate bufferClose the cap and you can add parafilm to secure the closurerecommended to do not subject sediment to freeze-thaw cycles in particular if the aim is to perform downstream analyses on sed-DNA extracts.Keep this sediment at -40°C or  -80°C for long term storage.        → On your notebook, note where the sediments are storedRepeat this process for all the sediments that will be extracted during this sessionAdd one volume of saturated phosphate buffer (0.12 M Na2HPO4; pH ≈ 8; stored less than one week at +4°C) for one volume of sedimente.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate bufferClose the cap and you can add parafilm to secure the closuree.g if you have weighed 0.5g of sediment, add 0.5mL of saturated phosphate buffer
?. [Desorb DNA from mineral and organic particlesAdjust lysis conditions]
Agitate tubes at slow speed at  for
0 Room temperature
?. [Precipitate contaminants]
Centrifuge
Centrifuge: 10000 33, 10 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  to the columnOptional : to increase yield you can heat buffer SE at   Do 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  to the columnOptional : to increase yield you can heat buffer SE at Do 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
?. [Precipitate contaminants]
Add  of buffer SL3 and vortex for Incubate at   for Centrifuge
150 µl
[in a fridge]
Centrifuge: 11000 34, 1 min