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.
The protocol is essentially the same as described above. However,
fungi produce cells with large vacuoles and it is often necessary to
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.