Chapter 8 :DNA Extraction from Fungus Using Silica Column

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Fungal DNA extraction is a crucial step in various mycological research and diagnostic applications. The silica column method offers an efficient and reliable way to obtain high-quality DNA from fungal samples. This blog post will outline the general procedure for DNA extraction from fungi using a silica column.

Materials Required

  • Fungal sample (mycelium, spores, fruiting bodies)
  • Liquid nitrogen (optional, for tissue grinding)
  • Lysis buffer
  • Proteinase K
  • RNase A
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Silica columns
  • Collection tubes
  • Microcentrifuge
  • Mortar and pestle or homogenizer

Procedure

  1. Sample Preparation and Lysis:
    • Depending on the fungal sample type, you may need to grind it using a mortar and pestle or homogenizer. Liquid nitrogen can be used to freeze the sample before grinding, making it easier to disrupt cell walls.
    • Add lysis buffer to the ground or disrupted fungal sample. This buffer contains detergents and enzymes, such as Proteinase K, that break down cell walls, membranes, and proteins, releasing DNA into the solution.
    • Incubate the sample at an appropriate temperature to ensure complete lysis.
  2. RNA Removal:
  3. Add RNase A to the lysate to degrade RNA, which can interfere with downstream applications.
  4. DNA Binding to Silica Column:
  1. Add binding buffer to the lysate. This buffer creates conditions that promote DNA binding to the silica membrane within the column.
  2. Transfer the lysate to a silica column.
  3. Centrifuge the column to bind DNA to the membrane and remove impurities.
  4. Washing:
  1. Wash the silica column with wash buffer to remove any remaining contaminants.
  2. Centrifuge the column to remove the wash buffer.
  3. DNA Elution:
  1. Add elution buffer (low salt buffer or water) to the silica column.
  2. Centrifuge the column to elute the purified DNA into a collection tube.

Tips and Considerations

  • Use fresh and pure fungal cultures or samples for optimal DNA yield and quality.
  • Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
  • Optimize lysis conditions (incubation time and temperature) for the specific fungal species and sample type.
  • Carefully follow the manufacturer’s instructions for the silica column kit.

Applications of Extracted Fungal DNA

The DNA extracted using the silica column method can be used for various applications, including:

  • PCR (Polymerase Chain Reaction)
  • Genotyping
  • DNA sequencing
  • Fungal identification and taxonomy
  • Pathogen detection
  • Population genetics studies
  • Phylogenetic analysis

Conclusion

Silica column-based DNA extraction from fungi is a widely used and reliable technique in mycological research and diagnostics. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality fungal DNA for your downstream applications.

Lysis Buffer :

The lysis buffer is developed using 0.5% CTAB, 1% EDTA, 2.5% Tris base and 5% NaCl

Binding Buffer : A binding buffer is a reagent used to bind DNA to silica during DNA extraction. It helps to purify DNA from various samples, including blood, cells, and tissues. It contains Guanidine Hydrochloride ,Tris-HCl and EDTA (pH. 8.0)

Washing buffer :

DNA Wash Buffer (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)

Elution buffer :

(0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )

Fungus gDNA Extraction Protocol 

  • 1. Take 50-100 mg of fresh or stored fungus tissues and grind in liquid nitrogen to make fine powder using mortar and pestle. 
  • 2. Transfer it into a microfuge tube 
  • 3. Add 800 μl of Lysis Buffer to the tube and mix it well. Add 10 μl of RNAse A into the lysate before incubation to remove the RNA. 
  • 4. Incubate tissue lysate result from above step at 65 C for 45 min. Mix sample during incubation by inverting tube. 
  • 5. Add 700 μl Chloroform/ Isoamyl alcohol (24:1) to the same microfuge tube and vortex to mix. Centrifuge at 10,000 rpm for 5-10 min. 
  • 6. Carefully aspirate supernatant resulting supernatant to a new 2 mL microfuge tube, make sure not to disturb the pellet or transfer any debris. 
  • 7. Add 600 μl of Binding Buffer  to the resultant supernatant from above step and mix well with inverting tubes 20-25 times. 
  • 8. Transfer 650 μl lysate to the DNA spin column, and centrifuge at 12,000 rpm for 1 min. Discard the flow through liquid. 
  • 9. Repeat above step, until the entire sample has been processed and retain column for further processing. 
  • 10. Place the column into same collection tube. Add 650 μl of Wash Buffer .Centrifuge at 10,000 rpm for 1 min. Discard the flow through. 
  • 11. Repeat the above  
  • 12. Place empty DNA spin column, with the lid open into the same collection tube and centrifuge at 10,000 rpm for 2 min. 
  • 13. Place the column into a new sterile 1.5 ml eppendorf tube, add 30 μl preheated Elution buffer. Incubate at room temperature for 5 min. (perform this step twice 30 μl + 30 μl = 60 μl) 
  • 14. Centrifuge 10,000 rpm for 1 min to elute pure gDNA. The first elution normally yields 60-70 % of DNA bound. A second elution with another 30 μl buffer will yield another 20%of the DNA. 
  • Note: Elution volume may vary as per downstream process 
  • 15. Discard the Column, and save elute. Do not reuse binding columns or collection tubes. 

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