Mouse Tail (or organ) Biopsy DNA Extraction
Lysis Buffer Proteinase K 25 ml 1M Tris pH 8.0 (FC 50mM) 100 mg dry Proteinase K (Sigma #2308) 50 ml 0.25M EDTA (FC 25 mM) 4.75 ml H2O 10 ml 5M NaCl (FC 100mM) 0.25 ml 1M Tris pH 8.0 5 ml Triton X-100 (FC 1%) 5 ul 1M CaCl2 dH2O to 500 ml FC 20 mg / ml, store @ 4 deg. C (FC = Final Concentration) (A cloudy suspension forms with storage @ 4 deg. C, so mix well before use)
- Cut 1/2" - 3/4" of mouse tail. (Check with your institution's Animal Studies Committee for their recommendations as to how this procedure should be implemented).
- Add tail fragment (or tissue of the same size) directly to 500 ul of extraction buffer and add 50 ul of Proteinase K.
- Incubate overnight at 52o-55oC.
- Add 500 ul of Tris pH 8.0 equilibrated phenol. Mix vigorously, spin (12,000-14,000 RPM) at room temperature for 2 minutes.
- Remove aqueous phase to a fresh tube.
- To the aqueous phase add an equal volume of Chloroform / Isoamyl Alcohol (49:1). Mix vigorously. Spin at room temperature for 2 minutes. Remove aqueous phase to a fresh tube.
- To the aqueous phase add 250 ul 7.5M NH4OAc and 750 ul isopropanol. Mix well. Spin at room temperature for 5 minutes. Wash the pellet once with 70% ethanol.
- Dry the pellet using a speedvac.
- Resuspend the DNA in 400 ul 1X TE; add 2 ul RNAse A (10 mg / ml) and incubate overnight at 37oC.
- Store at 4oC. Cut 100 ul (approximately 10 ug).
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