MITOCHONDRIAL DNA ISOLATION

PROCEDURE

Grind in a mortar or a Waring blender with 5-7 volumes of buffer A per gram of tissue. Use MCE at 350 l / L, and if necessary, with 5 ml 1 M DIECA / L.
Squeeze through cheesecloth, two layers of Miracloth.
Centrifuge 10 minutes at 1000 g
Decant the supernatant and centrifuge 10 minutes at 15,900 g.
Resuspend each pellet in a few drops of buffer G with brush, blend, bring about 10 ml/50 g, 15 ml/75 g
Centrifuge 10 minutes at 1000 g; empty most of the pellets of turbulence to eliminate the spongy layer, combined.
Bring supernatant to 10 mM MgCl2 (100 ml 1M/10 l). Make up to 20 g DNase / ml (100 l 2mg/ml/10 ml).
60 min. 4 C.
Platform Underlay Buffer, 20 ml ml/10-15, always use 20 ml or more.
Centrifuge 20 minutes at 12,000 g.
Resuspend in small volume buffer brush shelf, rising to about 10 g. ml/50-100
Centrifuge 10 minutes at 15,900 g.
Pellets Resuspend in NN (lysis) buffer (4-5 ml/50-75 g).
Add SDS to 0.5% (250 l of 10% / 5 ml NN). Shake well.
Add Proteinase K to 100 g / ml (25 L of 20 NN mg/ml/5 ml). Mix gently.
60 min. 37 C.
Add an equal volume of 3:1 phenol-saturated water, a mixture of chloroform-isoamyl alcohol.Emulsifying ca. 5 min.
Centrufuge 10 minutes at 7000 g.
Collect the supernatant and repeat 17 to 18: 3 total removals.
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, drainage, add equal volume 70% ethanol, let stand 10 min, 10 min at 8000-9000 g, dry drainage. Pellets of dry vacuum, 30 min. Two small Corex tubes are better than one 30 ml Corex.
Add 100-500 l 0.1x NTE, 10 l of RNase mix. Normally the use of 500 l per 50 g of tissue.
Hydrate for 30 min., 37 C.