MITOCHONDRIAL DNA ISOLATION

MITOCHONDRIAL DNA ISOLATION

1. Grind in mortar and pestle or Waring blender with 5-7 volumes buffer A per g tissue. Use MCE at 350 l/L, and if necessary, with 5 ml 1 M DIECA/L.
2. Squeeze through cheesecloth, two layers of Miracloth.
3. 10 min at 1000 g
4. Decant supernatant and centrifuge 10 min at 15,900 g.
5. Resuspend each pellet in a few drops of buffer G with paint brush; combine; bring to about 10 ml/50 g, 15 ml/75 g.
6. 10 min at 1000 g; pour off most; swirl pellet to remove fluffy layer; combine.
7. Bring supernatant to 10 mM MgCl2 (100 l 1M/10 ml). Bring to 20 g DNase/ml (100 l 2mg/ml/10 ml).
8. 60 min. 4 C.
9. Underlay shelf buffer, 20 ml/10-15 ml; always use 20 ml or more.
10. 20 min at 12000 g.
11. Resuspend in small volume shelf buffer with brush; bring to about 10 ml/50-100 g.
12. 10 min at 15900 g.
13. Resuspend pellets in NN (lysis) buffer (4-5 ml/50-75 g).
14. Add SDS to 0.5% (250 l of 10%/5 ml NN). Swirl thoroughly.
15. Add proteinase K to 100 g/ml (25 l of 20 mg/ml/5 ml NN). Swirl gently.
16. 60 min. 37 C.
17. Add equal volume of 3:1 water-saturated phenol, chloroform-isoamyl alcohol mixture. Emulsify ca. 5 min.
18. 10 min at 7000 g.
19. Collect supernatant; repeat 17 and 18: 3 total extractions.
20. Final supernatant; add 0.1 volume 8 M Ammonium acetate; then add 2 volumes of absolute ethanol.
21. 60 min, -80 C; 10 min at 8000-9000 g; drain; add equal volume 70% ethanol; let sit 10 min; 10 min at 8000-9000 g; drain dry. Vacuum dry pellet, 30 min. Two small corex tubes are better than one 30 ml Corex.
22. Add 100-500 l 0.1X NTE, 10 l RNase mixture. Typically use 500 l per 50 g tissue.
23. Hydrate 30 min., 37 C.