Mouse Tail (or organ) Biopsy DNA Extraction

 

<center> <h2> Mouse Tail (or organ) Biopsy DNA Extraction</h2> </center>

 

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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)

1. 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).
2. Add tail fragment (or tissue of the same size) directly to 500 ul of extraction buffer and add 50 ul of Proteinase K.
3. Incubate overnight at 52^o -55^o C.
4. Add 500 ul of Tris pH 8.0 equilibrated phenol. Mix vigorously, spin (12,000-14,000 RPM) at room temperature for 2 minutes.
5. Remove aqueous phase to a fresh tube.
6. 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.
7. To the aqueous phase add 250 ul 7.5M NH₄ OAc and 750 ul isopropanol. Mix well. Spin at room temperature for 5 minutes. Wash the pellet once with 70% ethanol.
8. Dry the pellet using a speedvac.
9. Resuspend the DNA in 400 ul 1X TE; add 2 ul RNAse A (10 mg / ml) and incubate overnight at 37^o C.
10. Store at 4^o C. Cut 100 ul (approximately 10 ug).