Chapter 16 : DNA Extraction from Soil

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Soil is a complex ecosystem teeming with life, from microscopic bacteria and fungi to larger organisms like insects and worms. Each of these organisms leaves behind traces of their DNA, providing a genetic snapshot of the soil’s biodiversity. Extracting DNA from soil allows scientists to identify the organisms present, study their interactions, and monitor changes in the environment.

The Process of Soil DNA Extraction

DNA extraction from soil can be challenging due to the presence of various contaminants, such as humic acids and other organic matter, which can inhibit DNA amplification and analysis. However, several methods have been developed to overcome these challenges and successfully extract DNA from soil samples.

1. Soil Collection and Preparation:

The first step in soil DNA extraction is collecting a representative soil sample. This involves taking multiple subsamples from different locations within the sampling area and mixing them thoroughly. The soil sample is then dried and sieved to remove large debris.

2. Cell Lysis and DNA Extraction:

The next step is to break open the cells of the organisms present in the soil sample and extract their DNA. This is typically done using a combination of physical and chemical methods, such as bead beating and enzymatic digestion. Bead beating involves shaking the soil sample with small beads to disrupt the cells, while enzymatic digestion uses enzymes to break down cell walls and release DNA.

3. DNA Purification:

Once the DNA has been extracted, it needs to be purified to remove any remaining contaminants. This can be done using various methods, such as centrifugation, filtration, and precipitation. Centrifugation spins the sample at high speeds to separate the DNA from other components, while filtration uses a membrane to trap contaminants and allow DNA to pass through. Precipitation involves adding a chemical to the sample to cause the DNA to clump together and precipitate out of solution.

4. DNA Amplification and Analysis:

The final step is to amplify and analyze the extracted DNA. This is typically done using polymerase chain reaction (PCR), which makes millions of copies of specific DNA sequences. The amplified DNA can then be analyzed using various techniques, such as gel electrophoresis and sequencing, to identify the organisms present in the soil sample.

Applications of Soil DNA Extraction

Soil DNA extraction has a wide range of applications in various fields, including:

  • Agriculture: Identifying beneficial and harmful microbes in soil to improve crop yields and reduce pesticide use.
  • Environmental Monitoring: Assessing the impact of pollution and climate change on soil biodiversity.
  • Forensic Science: Identifying human and animal DNA in soil samples for criminal investigations.
  • Medical Research: Discovering new antibiotics and other drugs from soil microbes.

The Future of Soil DNA Extraction

As DNA extraction and analysis techniques continue to advance, soil DNA extraction is becoming increasingly accessible and affordable. This is opening up new possibilities for research and applications in various fields, leading to a better understanding of the complex world beneath our feet.

DNA extraction buffer composition (10 ml):

1 M Tris-HCL (pH 8.0) : 2 ml

0.5 M EDTA (pH 8.0 ) : 200 μl 

5 M NaCl, : 140 μl

10% SDS : 2 ml

10 % CTAB : 2 ml

1M Mannitol : 2 ml

Nuclease free water : 1.66 ml

10X Phosphate buffer saline composition

Dissolve the following in 800ml distilled H2O.

  1. 80g of NaCl
  2. 2.0g of KCl
  3. 14.4g of Na2HPO4
  4. 2.4g of KH2PO4

Adjust pH to 7.4.

Adjust volume to 1L with additional distilled H2O and Sterilize by autoclaving.

Protocol :

  1. Mix the soil sample (~ 1 gm) with 2 ml PBS and transfer the entire mixture into 50 ml falcon tube.
  2. Add 5ml of 120mM Phosphate buffer saline (PBS, pH 7.4 ) and incubate for 10 minutes at  4°C.
  3. Centrifuge the sample at 4000 rpm for 20 minutes at 4°C.
  4. Discard the supernatant and rewash the pellet with PBS buffer.
  5. After PBS wash suspend the pellet in 10 ml DNA extraction buffer .
  6. Add 10 μl of Rnase A  and 10 μl of proteinase K.
  7. Incubate  the suspension at  65°C for 1 hour.
  8. After incubation,  centifuge at 12000 rpm – 10 min, Transfer clear supernatant into a new micro centrifuge tube.
  9. Add equal volume of PCI (25:24:1) and centrifuge it at 12000 rpm for 10 minutes at 4°C.
  10. Repeat the step no 10 if required.
  11. After centifugation, Transfer clear supernatant into a new micro centrifuge tube and add equal volume of Chloroform: Isoamyl alcohol (24:1 freshly prepared).
  12. Repeat the step no 11 if required.
  13. Mix the aqueous fraction with 1/10th volume of 3M sodium acetate (pH 5.2) and 2 volume of 100% chilled ethanol / Isoprapanol and keep the tube at -80 °C for 30 minutes.
  14. Centrifuge the tube at 14000 rpm for 10 minutes at 4°C, after centrifugation discard the supernatant.
  15. Wash with 70 % ethanol and centrifuge at 14000 rpm for 10 minutes at 4°C , after centrifugation discard the supernatant.
  16. Dry the pellet and disslove it in 25 μl elution buffer.

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