May 30, 2022
DNA Extraction and Purification from Soil
- 1Newcastle University
Protocol Citation: Justin GD Byrne, James JN Kitson 2022. DNA Extraction and Purification from Soil. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61npzvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 11, 2022
Last Modified: May 30, 2022
Protocol Integer ID: 56800
Keywords: Soil, DNA Extraction, DNA Purification
Abstract
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.
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.
Materials
Buffer components
| A | B | C | D | E | F | G | |
| Component | Contents | Required | Chemical molarity | per L | per 100 mL | pH | |
| Lysis solution 1 | guanidine thiocyanate | 147 mM | 118.16 | 17.33 g | 1.73 g | 9.0 | |
| trisodium phosphate | 228 mM | 380.13 | 86.67 g | 8.67 g | - | ||
| sodium chloride | 26 mM | 58.44 | 1.5 g | 0.15 g | - | ||
| 1 M Tris HCl | 67 mM | - | 67 mL | 6.7 mL | - | ||
| 0.5 M EDTA | 27 mM | - | 53 mL | 5.3 mL | - | ||
| Lysis solution 2 | aluminium ammonium sulphate* | 90 mM | 453.33 | 40.8 g | 4.08 g | - | |
| SDS | 1.25 % | - | 12.5 g | 1.25 g | - | ||
| Protein flocculant | ammonium acetate | 5 M | 77.0825 | 385.41 g | 38.54 g | - | |
| Inhibitor flocculant 1 | aluminium ammonium sulphate* | 180 mM | 453.33 | 81.6 g | 8.16 g | - | |
| Inhibitor flocculant 2 | calcium chloride dihydrate | 204 mM | 147.01 | 30 g | 3 g | - | |
| Binding solution | guanidine HCl | 5.5 M | 95.53 | 525.42 g | 52.54 g | - | |
| Wash solution | EtOH | 80 % | - | - | - | - | |
| Elution buffer | Pure H2O or 1mM Tris | - | - | - | - | 8.0 |
*aluminium ammonium sulphate = aluminium ammonium sulphate dodecahydrate (CAS 7784-26-1), if using anhydrous powder, adjust the calculation above.
Adjust the pH of Lysis solution 1 to 9.0 with 5M HCl and bring to volume with ddH2O. This will probably require much less than 20 ml of HCl and should be mixed in the fume hood. All other components apart from Elution buffer are used at the pH of the mixture without modification. Sterilise all solutions in a suitable manner (autoclave or filter).
Safety warnings
Follow appropriate precautions for molecular laboratory work, including following the COSHH guidelines for the reagents in use.
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.
Sample Lysis
Sample Lysis
6m 30s
6m 30s
Add 2g of 1.0mm to 1.4mm diameter acid-washed garnet beads to a 5ml Eppendorf screw-cap tube
Add 2200μL of Lysis Solution 1 and vortex briefly.
Add 0.25g of sample to the tube, and shake briefly by hand to mix the contents.
Parafilm the lids of the tubes to prevent leaks.
Place in Geno/Grinder 2010 with appropriate adapters and shake at 1750 RPM for 2 mins
Equipment
Geno/Grinder 2010
NAME
Pulverizer and Cell Lyser
TYPE
SPEX CertiPrep™
BRAND
12605297
SKU
2m
Wait 30 seconds
30s
Grind again for an additional 2 mins at 1750 RPM
2m
Centrifuge at 1000xg for 30 seconds to remove liquid from the lids of tubes
1000 x g, 25°C, 00:00:30
30s
Add 800μL of Lysis Solution 2
Centrifuge at 4,000xg for 1 min at room temperature.
4000 x g, 25°C, 00:01:00
1m
Transfer the supernatant to a fresh 1.5ml tube - or Transfer 1ml and save 500μl.
Centrifuge at 10,000xg for 1 min at room temperature. Transfer 500μl of supernatant to fresh tube 1.5ml tube.
10000 x g, 25°C, 00:01:00
1m
DNA Purification
DNA Purification
18m
18m
Add 200 μl volume of Protein flocculant, vortex briefly, and incubate on ice for a minimum of 10 mins.
10m
Centrifuge at 10,000xg for 1 min at room temperature.
10000 rpm, 25°C, 00:01:00
1m
Transfer supernatant to fresh tube 1.5ml tube.
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
Add 200 μl of Inhibitor flocculant mastermix to each sample
Centrifuge at 10,000xg for 1 min at room temperature.
10000 rpm, 25°C, 00:01:00
1m
Transfer supernatant to fresh 5ml tube.
Add 1568μl of Binding Solution and invert several times to mix.
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, 25°C, 00:01:00
Equipment
EZ-10 Spin Column & Collection Tube
NAME
Consumables
TYPE
Bio Basic
BRAND
SD5005.SIZE.100
SKU
LINK
1m
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, 25°C, 00:05:00
Equipment
96 well DNA plate with membrane (960ul each well)
NAME
Consumables
TYPE
Bio Basic
BRAND
SD5007.SIZE.12
SKU
LINK
Equipment
Breathable Film
NAME
Plate Seal
TYPE
StarLab
BRAND
E2796-3005
SKU
LINK
Natural, opaque, porous self-adhesive seal. Allows effective gas exchange for cellular and bacterial cultivation, while preventing contamination.
SPECIFICATIONS
5m
Add 392 μl of Wash Solution, centrifuge at 10,000xg for 1 min at room temperature, discard flow-through.
10000 rpm, 25°C, 00:01:00
1m
Centrifuge at 10,000xg for 1 min at room temperature, replace collection tube with a fresh 1.5ml tube.
10000 rpm, 25°C, 00:01:00
1m
Add 313μl of Elution buffer heated to 70°C directly to the silica filter membrane. Leave for 2 min at room temperature.
2m
Centrifuge at 10,000xg for 1min at room temperature.
10000 rpm, 25°C, 00:01:00
1m
Purified DNA is now in solution in the collection tube.