Mitochondrial DNA Isolation from Somatic Embryogenic Cell Cultures of Larix (Excerpt from Thesis).

Mitochondrial DNA is isolated by a modification of the methods described by Wilson and Chourey (1984) and Radetzky (1990).

Cell cultures at four days post-subculture are ground at 4 degrees Celsius in a mortar and pestle with sand and five volumes/gram fresh weight extraction buffer (0.4M mannitol, 1mM ethylene glycol-bis[aminoethyl ether] N',N',N',N', -tetraacetic acid (EGTA), 20mM N-[2-hydroxyethyl]piperazine-N' -[ethanesulfonic acid] (HEPES), pH 7.5, 0.1% bovine serum albumin, 0.05% cysteine, 10mM diethyldithiocarbamic acid (DIECA), 0.5% Polyclar AT (BDH Chemicals Ltd., England).

After filtration through two layers of Miracloth the homogenate is centrifuged at 150g for 10 minutes. The supernatant is then centrifuged three times at 3000g for 10 minutes to separate cellular debris, nuclei and proplastids from the mitochondria. Mitochondria are pelleted at 10000g for 20 minutes, resuspended in DNase buffer (0.4M mannitol, 20mM HEPES, pH 7.5, 10mM magnesium chloride) and treated with DNase I (0.1mg/mL) at 4 degrees Celsius for one hour.

After washing the mitochondria with DNase inhibiting buffer (100mM EGTA, 0.4M mannitol, 20mM HEPES, pH 7.5) mitochondria are further purified by centrifugation in a discontinuous Percoll gradient (45%, 21%, 14% Percoll) at 15000g for 15 minutes. The mitochondria that band are pooled and diluted with resuspension buffer (0.4M mannitol, 20mM HEPES, pH 7.5, 10mM EGTA, 10mM DIECA) and centrifuged several times at 10000g for 10 minutes to remove Percoll.

The washed and pelleted mitochondria are resuspended in lysis buffer (0.1M Tris.HCl, pH 8.0, 0.05M EDTA, 0.1M NaCl, 1% w/v sodium dodecyl sulfate, 1% w/v sarcosyl) and incubated at 65 degrees Celsius for 30 minutes. Organic material is removed by addition of 5M potassium acetate with incubation on ice for 20 minutes.

After centrifugation the supernatant is mixed with an equal volume of isopropanol. Preciptated DNA is dissolved in TE buffer (10mM/1mM).

DNA is further purified by extraction with phenol (buffered with TE), followed by two extractions with chloroform:isoamyl alcohol (24:1 v/v), reprecipitated and dissolved in TE buffer. RNA is removed by addition of RNase during the restriction endonuclease digestion of the mitochondrial DNA.

Notes:

1. DIECA must be added just prior to use.
2. Buffers and plasticware are autoclaved prior to use.
3. Perform DNA extraction in sterile flow hood for best results.
4. Percoll gradient steps can be eliminated without much loss in purity.
5. Restriction digestion of Larix mitochondrial DNA followed by agarose gel electrophoresis resulted in a complex banding pattern with bands of varying stoichiometry.