MITOCHONDRIAL?DNA?ISOLATION

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.

Squeeze through cheesecloth, two layers of Miracloth,10 min at 1000 g.

Decant supernatant and centrifuge 10 min at 15,900 g.

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.

10 min at 1000 g;pour off most;swirl pellet to remove fluffy layer;combine.

Bring supernatant to 10 mM MgCl2 (100 l 1M/10 ml).Bring to 20 g DNase/ml (100 l 2mg/ml/10 ml).60 min,4 C.

Underlay shelf buffer,20 ml/10-15 ml;always use 20 ml or more. 20 min at 12000 g.

Resuspend in small volume shelf buffer with brush;bring to about 10 ml/50-100 g.10 min at 15900 g.

Resuspend pellets in NN (lysis) buffer (4-5 ml/50-75 g).

Add SDS to 0.5% (250 l of 10%/5 ml NN).Swirl thoroughly.

Add proteinase K to 100 g/ml (25 l of 20 mg/ml/5 ml NN).Swirl gently.60 min.37 C.

Add equal volume of 3:1 water-saturated phenol,chloroform-isoamyl alcohol mixture.Emulsify ca. 5 min.10 min at 7000 g.

Collect supernatant;repeat 17 and 18: 3 total extractions.

Final supernatant;add 0.1 volume 8 M Ammonium acetate;then add 2 volumes of absolute ethanol.

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.

Add 100-500 l 0.1X NTE,10 l RNase mixture.Typically use 500 l per 50 g tissue.Hydrate 30 min,37 C.