TITLE: DNA Extraction and Purification from Soil
AUTHORS: Justingdbyrne  , James JN Kitson

[DESCRIPTION]
Affordable DNA extraction and purification from soil samples at scale. 

This protocol outlines an affordable DNA extraction and purification technique to be used with soil samples processed at scale. In the materials section, I provide details on how to make up reagents for each step. I provide the steps of the protocol in detail. The protocol steps follow the physical and chemical lysis of cells present in soils, the flocculation of various inhibitors of PCR, and the purification of extracted DNA by repeated centrifuging steps that bind and then elute DNA using a silica filter. 



[BEFORE_START]
The soil samples used in this work were collected using a soil auger from the upper 10cm of the soil core by a collaborator. Where possible, I removed soil from the centre of the core for analysis in order to minimise the potential for sample cross-contamination. 



[GUIDELINES]
When eluted in 1 mM Tris, the purified DNA can survive several freeze-thaw cycles. Depending on the samples analysed the purified DNA may benefit from additional dilution prior to PCR, especially if the end material is discoloured to a soil-brown.

[STEPS]
SECTION: Sample Lysis
1.  Add 2g of 1.0mm to 1.4mm diameter acid-washed garnet beads to a 5ml Eppendorf screw-cap tube


SECTION: Sample Lysis
2. Add 2200μL of Lysis Solution 1 and vortex briefly.
SECTION: Sample Lysis
3. Add 0.25g of sample to the tube, and shake briefly by hand to mix the contents.
SECTION: Sample Lysis
5. Place in Geno/Grinder 2010 with appropriate adapters and shake at 1750 RPM for 2 mins
 

SECTION: Sample Lysis
6. Wait 30 seconds
SECTION: Sample Lysis
7. Grind again for an additional 2 mins at 1750 RPM
SECTION: Sample Lysis
9. Add 800μL of Lysis Solution 2 
SECTION: Sample Lysis
10. Centrifuge at 4,000xg for 1 min at room temperature.
4000 x g, 1 min, 25 °C 

SECTION: Sample Lysis
11. Transfer the supernatant to a fresh 1.5ml tube - or Transfer 1ml and save 500μl.
SECTION: Sample Lysis
12. Centrifuge at 10,000xg for 1 min at room temperature. Transfer 500μl of supernatant to fresh tube 1.5ml tube.
10000 x g, 1 min, 25 °C 

SECTION: DNA Purification
13. Add 200 μl volume of Protein flocculant, vortex briefly, and incubate on ice for a minimum of 10 mins.
SECTION: DNA Purification
14. Centrifuge at 10,000xg for 1 min at room temperature.
10000 rpm, 1 min, 25 °C 

SECTION: DNA Purification
15. Transfer supernatant to fresh tube 1.5ml tube.
SECTION: DNA Purification
16. Make an Inhibitor flocculant master mix composed of:

    n x 110 μl of Inhibitor flocculant 1
and
    n x 110 μl of Inhibitor flocculant 2

Where n is the number of samples purified
SECTION: DNA Purification
17. Add 200 μl of Inhibitor flocculant mastermix to each sample
SECTION: DNA Purification
18. Centrifuge at 10,000xg for 1 min at room temperature.
10000 rpm, 1 min, 25 °C 

SECTION: DNA Purification
19. Transfer supernatant to fresh 5ml tube.
SECTION: DNA Purification
20. Add 1568μl of Binding Solution and invert several times to mix.
SECTION: DNA Purification
21. Fill a silica spin column to capacity with the above mixture, centrifuge at 10,000xg for 1 min at room temperature, discard flow-through and repeat until all mixture has passed through the spin column.

10000 rpm, 1 min, 25 °C 


 


SECTION: DNA Purification
22. Add 392 μl of Wash Solution, centrifuge at 10,000xg for 1 min at room temperature, discard flow-through.

10000 rpm, 1 min, 25 °C 
SECTION: DNA Purification
23. Centrifuge at 10,000xg for 1 min at room temperature, replace collection tube with a fresh 1.5ml tube.
10000 rpm, 1 min, 25 °C 

SECTION: DNA Purification
24. Add 313μl of Elution buffer heated to 70°C directly to the silica filter membrane. Leave for 2 min at room temperature.
SECTION: DNA Purification
25. Centrifuge at 10,000xg for 1min at room temperature.

10000 rpm, 1 min, 25 °C 

SECTION: DNA Purification
26. Purified DNA is now in solution in the collection tube.
SECTION: Sample Lysis
8. Centrifuge at 1000xg for 30 seconds to remove liquid from the lids of tubes
 1000 x g, 30 s, 25 °C 

SECTION: DNA Purification
21.1. As an alternative, 96 well silica plates may be used for processing at scale. 

When using these, fill the column with 600μl of the above mixture and seal the plate with a breathable film.  Centrifuge at 4,000 xg for 5 minutes over a 2.2ml deep-well plate to collect the flow-though. Repeat until all mixture has passed through the spin column using a new breathable film each time the column if filled. 

4000 x g, 5 min, 25 °C 
 

 



SECTION: Sample Lysis
4. Parafilm the lids of the tubes to prevent leaks.