Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Different Methods of Soil DNA Extraction

Asmita Kamble Asmita Kamble
Harinder Singh Harinder Singh

Published: Jan 20, 2020 DOI: 10.21769/BioProtoc.3521 Views: 16870

Edited by: Amey Redkar Reviewed by: Mugdha Rajendra SabaleMaria Sinetova

download pdf Download PDF
ask Ask a question
Favorite
Cited by

Abstract

Soil is the major reservoir of microbial diversity. Only 1% of microbial diversity can be cultured while 99% is still not culturable. It is necessary to extract DNA from soil in order to explore the 99% microbial diversity, which will be useful to harness novel industrial enzymes and natural products. In the present study, six traditional and two kit-based methods were utilized to obtain total soil DNA from Garden soil. Quality (Absorbance ratio at A260/A230, A260/A280 nm) of the extracted DNA was assessed and quantity was analyzed using the BioTek Epoch Microplate spectrophotometer. Quality of DNA is one of the important factors that should be taken in to account for downstream applications such as PCR or cloning experiments.

Keywords: Soil DNA search Non-culturable search DNA extraction search Garden soil search

Background

Soil is the largest terrestrial reservoir of microbial bio-diversity which significantly balance the critical cycle of carbon, nitrogen, and phosphorous; along with maintaining plant health, structure and fertility of the soil. 1 g of soil harbors 1,00,000-10,00,000 different bacterial and archaeal species (Satyanarayana, 2017). However, only 1% of the microbial communities can be cultured in the laboratory conditions while 99% are still unexplored because they are non-culturable. Total soil DNA extraction potentially onsets the journey of revealing hidden microbial diversity (Robe et al., 2003; Fatima et al., 2011 and 2014; Lamizadeh et al., 2019).

Soil DNA extraction includes two major steps: microbial cell lysis followed by purification to get rid of inhibitory molecules of humic acid and fulvic acid. Cell lysis can be performed via physical, mechanical and chemical approaches; or a combination of all three methods. These methods involve the use of detergents such as Sodium Dodecyl Sulfate (SDS) along with heat treatment in buffers like Tris-HCl or sodium phosphate buffers, along with the introduction of chelating agents such Ethylenediaminetetraacetic acid (EDTA) to protect extracted DNA from DNases which are readily present in external environment. There are different components which add to the effectiveness of cell disruption such as enzymatic treatment with lysozyme, using strong chaotropic agents such as guanidium salts, physical and mechanical treatment such as using liquid nitrogen to grind soil samples, ultra-sonication, glass beads, Zirconia beads and bead-beating approach for cell lysis. Various combinations of these methods are utilized to improve the yield of isolated DNA. Purification steps are conducted after DNA extraction with the help of phenol:chloroform:isoamyl-alcohol method (PCI method), ethanol precipitation, precipitation via polyethylene glycol, isopropyl alcohol, and spin columns. Purification is a crucial step as it involves the removal of PCR and restriction digestion inhibitors such as humic and fulvic acid which are co-extracted along with DNA. Hence, effective extraction procedure followed by stringent purification are crucial steps for isolating DNA which can be used for better understanding of microbial biodiversity (Fatima et al., 2011; Bag et al., 2016).

The present study was aimed to obtain a high yield of total soil DNA from garden soil by using different conventional methods of extraction as well as soil DNA kits (HiPura soil DNA kit and Dneasy power soil DNA kit). A comparative analysis was performed in terms of purity and yield using BioTek Epoch Microplate spectrophotometer for absorbance measurements.

Materials and Reagents

  1. For soil collection and processing
    1. Sterile 50 ml Falcon (Tarson, catalog number: 546041)
    2. Sterile scapula
    3. Garden soil
    4. Ethanol
    5. Liquid Nitrogen

  2. For Extraction of total soil DNA by different methods
    1. 0.1 mm Zirconia beads (BioSpec, catalog number: NC0362415)
    2. 2.7 mm Glass beads (BioSpec, catalog number: 11079127)
    3. 50 ml sterile Falcon (Tarson, catalog number: 546041)
    4. 2 ml Eppendorf (Tarson, catalog number: 500020)
    5. 1.5 ml Microcentrifuge tube (Tarson, catalog number: 5000010)
    6. Filter 0.45 µm (Jsil)
    7. 1,000 μl Microtips (Tarson, catalog number: 5210010)
    8. 200 μl Microtips (Tarson, catalog number: 521020)
    9. Disodium Hydrogen Phosphate anhydrous (Na2HPO4) (Molychem, catalog number: QB4Q640403)
    10. Sodium Dodecyl Sulfate (SDS, CH3(CH2)11OSO3Na) (Affymetrix, catalog number: 18220)
    11. Polyvinylpyrrolidone [PVPP, (C6H9NO)n] (Amresco, catalog number: 0507)
    12. Sodium Chloride (NaCl, Fisher Scientific, catalog number: 27605)
    13. Chloroform (CHCl3) (Molychem, catalog number: 13620)
    14. Sodium acetate trihydrate (CH3COONa·3H2O) (Fisher Scientific, catalog number: 1390M)
    15. Tris (Hydroxymethyl)Aminomethane Hydrochloride (Tris-HCl, C4H11NO3.HCl) (SRL, catalog number: 99438)
    16. Ethylenediaminetetraacetic acid (EDTA, Qualigens, catalog number: 12635)
    17. Phenol:Chloroform:Iso-amylalcohol 25:24:1 (PCI) (Sigma, catalog number: P2069)
    18. Sodium Dihydrogen orthophosphate Dihydrate (NaH2PO4·2H2O) (Molychem, catalog number: 25700)
    19. Cetyl trimethylammonium bromide (CTAB) (Molychem, catalog number: 22560)
    20. Mannitol (HiMedia, catalog number: RM9914)
    21. Lysozyme (Sigma, catalog number: 89833)
    22. Proteinase K (Sigma, catalog number: P2308)
    23. Sodium Lauroyl Sarcosine (Amersco, catalog number: 0719)
    24. Isopropyl alcohol (Molychem, catalog number: 1750)
    25. Iso-amyl alcohol (SRL, catalog number: 69931)
    26. Guanidine Hydrochloride (Sigma, catalog number: G3272)
    27. Glycine (MP Biomedicals, catalog number: 194825)
    28. Sodium Hydroxide (Molychem, catalog number: 25800)
    29. Polyethylene Glycol (6000) (LOBA, catalog number: Art.5312)
    30. Hydrochloric acid (Molychem, catalog number: 23540)
    31. RNase (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    32. Proteinase K (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    33. Spin Column (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    34. Wash buffer1 (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    35. Wash buffer2 (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    36. Elution buffer (Invitrogen PureLink Genomic DNA mini kit, catalog number: K1820-01)
    37. Ethanol 99.9% pure (Changshu Hongsheng Fine Chemicals, catalog number: 1170)
    38. Stock solution (see Recipes)
      1 M NaCl
      1 M NaH2PO4
      1 M Na2HPO4
      0.2 M Na2HPO4
      1 M Tris-HCl
      0.2 M EDTA
      1 M HCl
      50x TAE buffer
    39. Working stock solution (see Recipes)
      DNA extraction buffer 1
      50% PEG (6000)
      0.6 M NaCl
      Chloroform:Isoamyl alcohol (24:1)
      3 M sodium acetate
      T10E1 buffer
      PCI
      0.1 M Phosphate buffer saline
      DNA extraction buffer 2
      10% CTAB + 0.7 M NaCl
      TEN buffer (pH 8.0)
      TEN buffers (1 ml) supplied with 0.2 mg Lysozyme
      20% SDS
      T50E1
      Lysozyme (10 mg/ml)
      Guanidine-HCl (5 M)
      10% Sodium Lauryl Sarcosine
      TENP
      Lysozyme solution
      SDS solution
      1x TAE buffer
      70% Ethanol

  3. Commercial kits for soil DNA extraction
    1. HiPurA soil DNA kit (HiMedia, catalog number: MB542)
    2. Dneasy Power Soil (Qiagen, catalog number: 12888-50)

  4. For agarose gel electrophoresis
    1. Tris-Base (MP Biomedicals, catalog number: 103133)
    2. EDTA (Qualigens, catalog number: 12635)
    3. Glacial Acetic acid (Molychem, catalog number: 21020)
    4. Ethidium bromide (Sigma-Aldrich, catalog number: E7637)
    5. 1 kbp ladder (Generuler, catalog number: SM0313)
    6. 6x gel Loading dye (Frementas, catalog number: R0611)
    7. Agarose (MP Biomedicals, catalog number: 218072090)

Equipment

  1. Sieve (Mesh size of 0.22 mm, Sumeet Royal Interchangeable sieve)
  2. Mortar and pestle (diameter of Mortar: 6 cm)
  3. Pipettes 100-1,000 μl, 20-200 μl, 0.5-10 μl, 2-20 μl (Glison)
  4. Water bath (Metalab, model: MSI14)
  5. Centrifuge (Eppendrof, model: 5424R)
  6. Micro-centrifuge (Pfact, model: 5804K)
  7. Incubator Shaker (Hexatech, model: HIPL-035C)
  8. Ice-machine (Wensar, model: LMIF series)
  9. -20 °C refrigerator (Thermo)
  10. Refrigerator (LG, model: GL-365YVQG5)
  11. Sonicator (Cole-Parmer, model: 08895-22)
  12. Weighing machine (Shimadzu, model: AUX220)
  13. Horizontal gel electrophoresis apparatus (Techno Source, model: Sleek Gel)
  14. Gel Documentation system (Bio-Rad, model: Universal Hood II)
  15. Epoch Microplate spectrophotometer with Take3 plate accessories (BioTek, serial No.: 401554)
  16. Microwave (LG microwave, model: MS-2347BS)

Software

  1. Gen-5 Data Analysis Software (version 3.03)
  2. Image Lab version 5.2.1 build11

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial genetics > DNA

Environmental science > Bacterium > Isolation

Molecular Biology > DNA > DNA extraction

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A