wetbench author created giovannellilab

Genomic DNA Extraction from shark skin

Extraction of genomic host DNA from a piece of shark skin preserved frozen.

Duration: 2.5h + overnight DNA precipitation +1h

Adapted from: Donato Giovannelli sediment DNA extraction with suggestions from Sergio Stefanni

Materials

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

Equipment

  • bench-top microcentrifuge up to 16.000 xg
  • vortex
  • Benchtop shaking incubator @ 52 °C
  • Chemical Hood
  • Pipets P1000 and P200

Solutions

  • Extraction buffer EB : (100 mM Na2HPO4 pH 8, 100mM tris-HCL pH 8, 100 mM EDTA pH 8, 1,5 M NaCl, 1% CTAB )
  • 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 Buffer

Procedure

  1. Dry the skin
  2. Put the piece of skin on an aluminum foil, then scrape and fray it with a surgical blade
  3. Put the piece of skin in a clean stearilized 2ml tube
  4. Add 850 µl of EB (extraction buffer)
  5. Add 100 µl of Lysozime (100 mg/ml)
  6. Add 5 µl of Proteinase K (20 mg/ml)
  7. Add 50 µl of SDS
  8. Invert tube
  9. Incubate in a benchtop shaking incubator @ 52°C overnight
  10. Vortex 1’
  11. Centrifuge 3’ @ 14,000 rpm.
  12. Recover supernatant
  13. Add 1 vol (ca. 1 ml) of Phenol:Chl:IAA
  14. Invert 100 times or until emulsion forms
  15. Centrifuge 3’ @ 14,000 rpm. Recover aqueous phase (generally the top phase) to clean 2 ml tube
  16. Add 1 vol Chl:IAA. Invert 100 times or until emulsion forms.
  17. Centrifuge 3’ @ 14,000 rpm. Recover aqueous phase (generally top phase) to clean 2 ml tube
  18. Add 0.1 vol of Na-Acetate and 0.7 vol of Isopropanol
  19. Invert several times
  20. Refrigerate @ -20 °C for 1h 20’
  21. Centrifuge 30’ @ 14,000 rpm. Carefully remove supernatant
  22. Add 0.5 ml of EtOH 70% ice cold and invert tube several times
  23. Centrifuge 5’ @ 14,000 rpm. Carefully remove supernatant. Air dry for ca. 20’ @ 37 °C with the Eppendorf open
  24. Resuspend in 50 µl of tris buffer (C6)
  25. Run 3 µl on 1- 1.5% agarose gel to check the presence of gDNA and preserve @ -20

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

  • It’s possible that the 20’ at @ 37°C are not enough to dry the sample; after 20’ check the sample and maybe wait 10’ more
  • If the skin sample is very think, remove the outside skin and/or cut it in smaller pieces

Calculations

none