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