MITOCHONDRIALDNAISOLATION

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 ℃.

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 ℃.

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 ℃.