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Welcome to The Science Advisory Board's extensive database of research protocols for the biomedical sciences. Protocols are organized by techniques and are fully searchable by key word(s). Protocols are divided into a brief description of the methods, a step-by-step overview of the procedure, a collection of recipes, the supplies needed, and any helpful tips. Members submitting protocols will be rewarded through the ViewPoints system.
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MITOCHONDRIAL DNA ISOLATION

Posted on Friday, November 07, 2003

Description
MITOCHONDRIAL DNA ISOLATION

Procedure
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.


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