Purification Of Mitochondrial Dna

The basic procedure for breaking cells with glass beads as described in the reference Lang et al. (1977) is as follows.


 

MtDNA from Protists

A minimum of 2-3 g wet weight of cells are necessary for one DNA extraction (preferably 10-20 g). The cells are harvested in the early stationary phase by centrifugation. After resuspension in a sorbitol buffer (0.6 M sorbitol, 5 mM EDTA, 50 mM Tris pH 7.4), the cells are broken mechanically by shaking with glass beads, and a crude mitochondrial fraction is isolated by differential centrifugation. The mitochondrial fraction is lysed in the presence of 1% SDS and 100 ug/ml proteinase K, at 50 degrees Celsius for 1 hr. SDS is eliminated from the lysate by addition of 1 M NaCl; after 1 hr on ice, the precipitate (SDS protein complex) is removed by centrifugation. The total nucleic acids are fractionated on a CsCl gradient (1.1 g/ml, 40 000 rpm for 48 hours) in the presence of 10 ug/ml bis-benzimide (Hoechst 33258, Serva). The upper band (A+T- rich DNA) in the gradient consists in many instances of mtDNA, and is extracted and re-centrifuged in one or two subsequent CsCl gradients. A final yield of 0.3 – 5 ug mtDNA can be expected.


 

MtDNA from Fungi

The protocol is essentially the same as described above. However, many fungi produce cells with large vacuoles and it is often necessary to start from 10-30 g wet weight cells in order to obtain a few ug of mtDNA. 
When a fungus produces mycelia it can be alternatively ground in liquid nitrogen and the total nucleic acids extracted with guanidine hydrochloride (Deeley et al., 1977), except that we use a 6M guanidine-HCl solution for the lysis.

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