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DNA Extraction from Sediments or Soils

Extracting total high molecular weight DNA from sediments and soils samples. The protocols with small modifications can be applied to other solids such as rocks and minerals.

Duration: 2.5 h + variable DNA precipitation step

Adapted from: Vetriani et al., 1999 AEM

Materials

  • 2 ml Eppendorf tube (a total of 5 for each samples)
  • 1.5 ml Eppendorf tube (one for each samples)

Equipment

  • Bench-top Microcentrifuge up to 16.000xg
  • Vortex
  • Dry incubator @ 37 ˚C
  • Water bath warmed @ 65˚C
  • Chemical Hood
  • Pipets P1000 and P200

Solutions

  • Extraction buffer EX (50mM Tris-HCL pH 8.0, 20 mM EDTA pH 8.0, 100 mM NaCl)
  • SDS 20%
  • Lysozime 100 mg/ml
  • Proteinase K 20 mg/ml
  • Phenol:Chloroform:Isoamyl alcohol 25:24:1
  • Chloroform:Isoamyl alcohol 24:1
  • Isopropanol 100%
  • Sodium Acetate 3M
  • Ethanol 70% ice cold
  • Tris-HCl buffer pH 8.0 50mM

Procedure

  1. Add 850 µl of extraction buffer EB to a clean sterilized 2 ml tube
  2. Add ca. 0.8 g of sediment
  3. Add 100 µl of Lysozime [100 mg/ml]. Incubate shaking @ 37 ˚C for 30’
  4. Add 5 µl of Proteinase K [20 mg/ml]. Incubate shaking @ 37˚C for 30’
  5. Vortex for 1’
  6. Add 50 µl of 20% SDS. Incubate 1 h @ 65 ˚C in waterbath inverting every 15-20’
  7. Vortex for 1’
  8. Centrifuge 3’ @ 14,000xg. Recover supernatant in clean 2 ml tube
  9. Add 350 µl of EB buffer to the sediment pellet, invert ca. 50 times and centrifuge again. Recover supernatant in previous tube
  10. Add 1 vol (ca 1 ml) of Phenol:Chloroform:IAA. Invert 100 times or until emulsion forms
  11. Centrifuge 3’ @ 14,000xg. Recover aqueous phase (generally top phase) to clean 2 ml tube
  12. Add 1 vol of Chloroform:IAA. Invert 100 times or until emulsion forms
  13. Centrifuge 3’ @ 14,000xg. Recover aqueous phase (generally top phase) to clean 2 ml tube
  14. Add 0.1 vol of Na-Acetate and 0.7 vol of Isopropanol. Invert several time and incubate. Incubation can be carried out overnight @ Room Temperature or +4°C or for a shorter amount of time (2h @ -20°C) depending on the sample and expected output
  15. Centrifuge 30’ @ 14,000xg @ 4°C. Carefully remove supernatant
  16. Add 0.5 ml of EtOH 70% ice cold and Invert tube several times
  17. Centrifuge 5’ @ 14,000xg. Carefully remove supernatant. Air dry for ca. 20’ or speedvac for 5’
  18. Resuspend in 50 µl of Tris buffer or PCR grade water and transfer in clean 1.5 ml tube
  19. Run 3 µl on 1-1.5% agarose gel to check presence of high quality DNA and preserve the rest of the DNA @ -20 ˚C clearly labelled

Expected results

Expect a clear high molecular weight band (~20 kb or higher) from each sample. A small amount of DNA degradation in the form of a smear can be expected. Reduce vortexing and pipetting steps if this becomes excessive

Common problems, troubleshooting and solutions

  • Phase inversion during the phenol:chloroform extraction step (step 11) might happen in samples with high salinity or oin the presence of compounds that can change the aqueous phase density
  • Excessive degradation of the DNA can happen with certain samples, If this is due to the extraction procedure (and not by sample collection or preservation) you can reduce it by reducing vortexing and lysis steps
  • In case of low or no DNA is extracted, it is possible to extract in parallel from multiple aliquots of the same sample and combine the different pellets during step 18 using the same volume to resuspend multiple DNA pellets
  • The final DNA might still contain inhibitors. These can be removed in different ways, depending on the amount of DNA available and the intended use

Calculations

none

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