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Fishery Bulletin 88(3), 1990 



Density-gradient ultracentrifugation 



The cornerstone of our isolation method is low-volume 

 ultracentrifugation using the Beckman TL-100 Table- 

 top Ultracentrifuge. The short rotor radius and high 

 available g-forces of this product serve to significant- 

 ly reduce spin times over current practices. The main 

 requirement is simply to "downsize" existing protocols 

 to be compatible with 3-mL centrifuge tubes. Isolation 

 of mtDNA using the TL-100 appears to be less labor- 

 intensive as well as faster in producing highly purified 

 mtDNA than existing techniques. 



Ultracentrifugation was accomplished at approx- 

 imately 200,000 xg (70,000 rpm) overnight (15-17 

 hours) at 4°C in a fixed-angle rotor (Beckman TLA- 

 100.3) with six positions. Deceleration was set at 5. 

 Following ultracentrifugation the mtDNA bands, which 

 are visible in fluorescent light 0.5-0.7 mm below the 

 nuclear DNA bands (Fig. 1), were withdrawn using an 

 18-gauge stainless-steel hypodermic needle inserted 

 through the top of the tube to a position slightly below 

 the mtDNA band. Withdrawn sample volume was then 

 adjusted to 0.5 niL with STE and extracted several 

 times with NaCl-saturated n-butanol at room tempera- 

 ture to remove the ethidium bromide (Lansman et al. 

 1981). 



Following butanoi extraction, the mtDNA was 

 ethanol-precipitated using essentially the procedure of 

 Maniatis et al. (1982). Sample molar concentrations 

 were adjusted by dilution with 0.33 niL of cold, sterile, 

 distilled H2O. Exactly two volumes of cold ethanol 

 were added to the solution which is briefly vortexed. 



Figure 1 



Appearance of nuclear and mitochondrial DNA bands in UV light 

 after 17 hours of ultracentrifugation in CsCl/ethidium-bromide gra- 

 dients at 200,000 X g and 4°C: upper bands are nuclear DNA, lower 

 bands mtDNA. Material at the top of the tubes is the "skin" which 

 forms during ultracentrifugation. Starting tissue was 1 -2.5 g of ripe 

 ovary from Spanish sardine Sardinella aurita. 



The sample is stored overnight at -20°C after which 

 mtDNA is pelleted by centrifugation in the fixed-angle 

 rotor in the Beckman TL-100 at 30,000 x g for 30 

 minutes at 2°C. After complete drying by evaporation 

 (no odor of ethanol remaining) the mtDNA pellet is 

 dissolved in 50 f^L of cold TE (Table 1) and stored in- 

 definitely at -20°C. 



Fluorometric quantification of mtDNA 



Yield quantification followed Paul and Myers (1982). 

 Standard curves were generated from fluorescence of 

 known DNA quantities in a 1.5x10"'* M solution of 

 Hoechst 33258 (CalBiochem Cat. #SR5A03-0388). 

 Fluorescence was measured using an Aminco J4-7439 

 Fluoro-Colorimeter (GE F4T4/BL UV lamp and an 

 R-136 phototube) at photomultiplier settings of 1, 3, 

 and 10. mtDNA concentrations were calculated using 

 the geometric mean regression of the standard curve. 

 Yields were determined as fig mtDNA per g of start- 

 ing tissue. 



[Restriction endonuciease digestion 



Immediately prior to digestion with restriction en- 

 zymes, samples were treated with RNAse-A to a con- 

 centration of 10 fxglmL and incubated at room temper- 

 ature for 1 hour. Samples were then heated to 70 °C 

 for 5 minutes and cooled on ice before addition of the 

 restriction enzymes. In this initial study only two six- 

 base-pair restriction endonucleases were used: £'co -RI 

 and Hind-Ul. Digestions were carried out according 

 to the reaction conditions specified by the manufac- 

 turers. Reaction mixtures contained 0.2-1.0 ^g of 

 mtDNA in a final volume of 20 ^L adjusted with 



