TITLE: Inhibitor-free DNA extraction from soil and sediment samples
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes how to extract inhibitor-free DNA from soil and sediment samples. 5 g of soil or up to 10 g of sediment can be processed in one extraction, but there is also a miniaturized version for 250 mg of input material, if less DNA is required. The protocol is based on the DNeasy PowerMax Soil Kit but costs much less. A lot of the buffers can be found in the following patent https://patents.google.com/patent/US7459548B2/en


[BEFORE_START]
Make sure all buffers are prepared before starting.

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.  

[STEPS]
SECTION: Protocol for up to 10 g of input material
1. Prepare one 50 mL centrifuge tube per sample with 15 g of garnet beads.
  

SECTION: Protocol for up to 10 g of input material
2. Add up to 10 g of soil to the tube.
  

 

SECTION: Protocol for up to 10 g of input material
3. Add 15 mL  and 1.2 mL. Vortex shortly. 


SECTION: Protocol for up to 10 g of input material
4. Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 10 min .
  
 


SECTION: Protocol for up to 10 g of input material
5. 2500 x g, 3 min, 20 °C . Transfer the supernatant to a new tube.
  
 



SECTION: Protocol for up to 10 g of input material
6. Add 5 mL , vortex shortly, and incubate at 4 °C for 10 min .

SECTION: Protocol for up to 10 g of input material
7. 2500 x g, 4 min, 20 °C . Transfer the supernatant to a new tube avoiding the pellet. The solution may still be colored, depending on the input material. 
    

SECTION: Protocol for up to 10 g of input material
8. Add 4 mL . A precipitate may form. Vortex shortly, incubate at 4 °C for 10 min .
  

SECTION: Protocol for up to 10 g of input material
9. 2500 x g, 4 min, 20 °C . The solution will clear up. Avoiding the pellet, transfer up to 15 mL to a new tube.
  

SECTION: Protocol for up to 10 g of input material
10. Add 30 mL . Vortex or invert to mix.

SECTION: Protocol for up to 10 g of input material
11. Add the mixture to a maxi spin column (e.g. Epoch Life Science) in a 50 mL centrifuge tube.
  

SECTION: Protocol for up to 10 g of input material
12. 2500 x g, 30 s, 20 °C . Discard the flow-through. Repeat once to bind the complete sample volume.

SECTION: Protocol for up to 10 g of input material
13. Add 10 mL . 2500 x g, 5 min, 20 °C to wash and dry the column.

SECTION: Protocol for up to 10 g of input material
14. Transfer the column to a new tube. Add 1 mL . Incubate for 3 min  at Room temperature .

SECTION: Protocol for up to 10 g of input material
15. 2500 x g, 1 min, 20 °C to elute the DNA. DNA eluate should be completely colorless and ready to go for downstream analysis.
   

SECTION: Protocol for up to 250 mg of input material
16. Prepare one 2 mL centrifuge tube per sample with 750 mg of garnet beads.
SECTION: Protocol for up to 250 mg of input material
17. Add 250 mg of soil or sediment sample.

SECTION: Protocol for up to 250 mg of input material
18. Add 750 µL  and 60 µL. Vortex shortly.

SECTION: Protocol for up to 250 mg of input material
19. Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 10 min .

 

SECTION: Protocol for up to 250 mg of input material
20. 10000 x g, 3 min, 20 °C . Transfer the supernatant to a new tube.

SECTION: Protocol for up to 250 mg of input material
21. Add 250 µL , vortex shortly, and incubate at 4 °C for 5 min .
SECTION: Protocol for up to 250 mg of input material
22. 10000 x g, 1 min, 20 °C . Transfer the supernatant to a new tube.

SECTION: Protocol for up to 250 mg of input material
23. Add 200 µL . A precipitate may form. Vortex shortly, incubate at 4 °C for 5 min .
SECTION: Protocol for up to 250 mg of input material
24. 10000 x g, 1 min, 20 °C . Transfer 600 µL of the supernatant to a new tube.

SECTION: Protocol for up to 250 mg of input material
25. Add 1200 µL . Vortex to mix.

SECTION: Protocol for up to 250 mg of input material
26. Load 650 µL of the mixture to a mini spin column (e.g. Epoch Life Science).

SECTION: Protocol for up to 250 mg of input material
27.  10000 x g, 30 s, 20 °C . Discard the flow-through. Repeat two times to bind the complete sample volume.

SECTION: Protocol for up to 250 mg of input material
28. Add 500 µL . 10000 x g, 30 s, 20 °C to wash the column. Discard the flow-through.
SECTION: Protocol for up to 250 mg of input material
29.  10000 x g, 1 min, 20 °C to dry the column. Transfer the spin column to a clean 1.5 mL microcentrifuge tube.
SECTION: Protocol for up to 250 mg of input material
30. Add 50 µL . Incubate for 3 min  at Room temperature .

SECTION: Protocol for up to 250 mg of input material
31. 10000 x g, 1 min, 20 °C to elute the DNA. DNA eluate should be completely colorless and ready to go for downstream analysis.