DNA Extraction
Genomic DNA extraction from tails or other tissue
8. 3. 08. BumSup Kwon
- Lysis buffer (sterilize the solution by filtration)
20 mM Tris-cl (pH 8.0) 5 mM EDTA (pH 8.0) 400 mM NaCl 1 % SDS
- Proteinase K (20 mg/ml)
- phenol:chloroform:isoamyl alcohol mixture (25:24:1)
1. Prepare 0.5 ml of lysis buffer per each sample and add proteinase K (final conc 400 ug/ml).
2. Incubate tissue (100 mg) in the lysis buffer (0.5 ml) overnight at 55°C in a shaking water bath.
3. Add an equal volume (0.5 ml) of phenol:chloroform:isoamyl alcohol mixture, and then mix by shaking for 10 min.
4. Centrifuge at 12,000 rpm for 5 min, and then transfer the upper aqueous phase to a new tube.
5. Precipitate DNA by adding an equal volume (0.5 ml) of isopropanol.
6. Centrifuge at 12,000 rpm for 15 min, and then discard the isopropanol.
7. Rinse the pellet with 1 ml of 70% ethanol.
8. Centrifuge at 12,000 rpm for 5 min, and then discard the 70% ethanol.
9. Dry the pellet, and then dissolve it with 100-200 ul of dH2O or TE buffer.