Add 720 µL of ATL buffer from the DNeasy Blood & Tissue Extraction Kit (Qiagen)
Vortex at full speed for 5 min.
Transfer the mixture to a 2 mL tube containing 20 µL of Proteinase K
Mix by pipetting
Incubate 2 hours at 56°c
Centrifuge 2 min at 11,000 x g at 4°C
Transfer the clear supernatant to a new 2 mL tube
Add 150 µL of buffer SL3 and vortex 5 s.
Incubate 5 min at 0-4°C
Centrifuge 1 min at 11,000 x g at 4°C
Place the NucleoSpin® Inhibitor Removal Column (red ring) onto a collection tube (2 mL, lid)
Load up to 650 µL of the clear supernatant from step 3 onto the filter and centrifuge 1 min at 11,000 x g (repeat this step as many time as there is still some supernatant from step 3 to be filtered. After each centrifugation, collect the filtered liquid in a clean tube (several collecting tubes are required, but only 1 column for all the 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 ride of this pellet, and continue with the clear supernatant.
Adjust binding conditions (step 6 from the NucleoSpin® Soil (MACHEREY-NAGEL GmbH & Co., Düren Germany)
Place a NucleoSpin® Soil Column (green ring) in a Collection tube (2 mL)
Load 550 µL of sample onto the column
Centrifuge 1 min at 11,000 x g
Discard the flow through and place the column back in a collection tube
Load the remaining sample onto the column
Centrifuge for 1 min at 11.000 x g
Discard the flow through and place the column back into the collection tube
Wash and dry silica membrane
Note: the same collection tube is used throughout the entire washing procedure to reduce plastic waste
-Add 500 µL of Buffer SB to the NucleoSpin® Soil Column
-Centrifuge for 30 s at 11.000 x g
-Discard the flow through and place the column back into the collection tube
-Add 550 µL of Buffer SW1 to the NucleoSpin® Soil Column
-Centrifuge for 30 s at 11.000 x g
-Discard the flow through and place the column back into the collection tube
-Add 650 µL of Buffer SW2 to the NucleoSpin® Soil Column
-Close the lid and vortex for 2s.
-Centrifuge for 30 s at 11.000 x g
-Discard the flow through and place the column back into the collection tube
-Add 650 µL of Buffer SW2 to the NucleoSpin® Soil Column
-Close the lid and vortex for 2s.
-Centrifuge for 30 s at 11.000 x g
-Discard the flow through and place the column back into the collection tube
Note: if for any reason, the liquid in the collection tube has touched the NucleoSpin® Soil Column after the drying step, discard flow through and centrifuge again
Place the NucleoSpin® Soil Column into a new microcentrifuge tube (not provided in the kit)
Add 50 µL of Buffer SE previously heated to 37°C to the column
Do not close the lid and incubate for 1 min 30 s at room temperature (18-25°C)
Close the lid and centrifuge for 30 s at 11.000 x g
Repeat the same steps with the same column and the same collection tube to get a final extract of 100 µl: Add 50 µL of Buffer SE previously heated to 37°C to the column
Do not close the lid and incubate for 1 min 30 s at room temperature (18-25°C)
Close the lid and centrifuge for 30 s at 11.000 x g
Throw the column and keep the tube containing the 100 µL of DNA
Store DNA frozen at -20°C (or -80°C for longer storage).