© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 1 185 A rapid miniprep method for the preparation of yeast mitochondrial DNA A.Defontaine, F.M.Lecocq and J.N.Hallet* Laboratoire de Cytophysiologie Vegetale, University de Nantes 2, rue de la Houssiniere, 44072 Nantes Cedex 03, France Submitted November 14, 1990 Two protocols have been recently proposed to speed up the preparation of Saccharomyces cerevisiae mitochondrial DNA (mt DNA). The first (1) is a simplified procedure derived from pre- existing methods using a CsCl gradient, while the other (2) is based on the isopycnic purification of the mitochondrial fraction from lysed spheroplasts. Both the above procedures require an ultracentrifugation step. We present here a rapid, simple and inexpensive miniprep method which requires no more than usual bench equipment and which gives reliable results for the routine analysis of mtDNA from both wild and laboratory strains of 5. cerevisiae. For each strain, the extraction of mtDNA was performed from an overnight 20 ml culture in YPD (1% yeast extract, 2% peptone, 2% dextrose) at 28°C under shaking. The cells were harvested by centrifugation at 500 Xg for 5 min and the pellet, which corresponds to 0.3—0.4 g (wet weight) was washed twice in sterile distilled water and then once in 1.2 M sorbitol, 50 mM EDTA, 2% mercaptoethanol. The pellet was then resuspended in 5 ml of Sol A (0.5 M sorbitol, 10 mM EDTA, 50 mM Tris, pH 7.5) containing 2% mercaptoethanol and 0 . 2 - 1 mg/ml of zymolyase 20 T, and then incubated at 37°C for 45 min under gentle agitation. During this step the majority of the formed spheroplasts were osmotically lysed. The suspension can be sonicated using an ultrasonic cell disruptor (Bioruptor UCD 130, Tosho Denki Co Ltd) at 19.3-19.5 KHz for 1 to 5 min (until the sample becomes visually more clear). This step, which breaks open the unlysed protoplasts, was not absolutely necessary but did improve the final result. The cellular lysate was then transferred into microfuge tubes and centrifuged at lOOOxg for 10 min. The supernatant containing the mitochondria was centrifuged at 15000xg for 15 min and the crude mitochondria! pellet was carefully washed three or four times with Sol A to eliminate genomic DNA contamination (this step is crucial). The mitochondria were resuspended in 0.2-0.4ml of Sol B (100 mM NaCl, 10 mM EDTA, 1% Sarkosyl, 50 mM Tris, pH 7.8) and allowed to lyse for 30 min at room temperature. The nucleic acids were then purified from the mitochondrial lysate by extraction with phenol-chloroform and ethanolic precipitation. RNAs could be further degraded by RNAse A. Good yields were obtained (10 to 20 /tg DNA per 0.3-0.4 g of wet weight) and the mt DNA was pure enough (absorption ratio at 260/280 nm = 1.8-1.9) for RFLP analysis (fig. 1). This protocol is efficient for many yeast strains and should take no more than 4 - 6 hours. It is cheap and easy to perform (since it avoids ultracentrifugation steps and the use of carcinogenic dyes) and can be adapted for obtaining much larger amounts of mtDNA. It is suitable for studying the polymorphism of a large number of wild strains or for strain identification. REFERENCES 1 Gargouri.A. (1989) Curr. Genet. 15, 235-237. 2. Querol.A. and Barrio.E. (1990) Nucl. Acids Res. 18, 1657. 2 3 Figure 1. Agarose gel electrophoresis of EcoRV digested mtDNA of Saccharomyces cerevisiae strains. Lanes 2 - 3 : industrial enological strains EG 8 and 522 Davis; lanes 4 - 5 wild strains; lanes 7 and 8: undigested and digested mtDNA of the laboratory strain S 288 C; lane 7, L:L dsRNA; lanes 1 and 6: HindUl restriction fragments of Lambda DNA and HaeUl restriction fragments of PhiX DNA. • To whom correspondence should be addressed

A rapid miniprep method for the preparation of yeast ......Two protocols have been recently proposed to speed up the preparation of Saccharomyces cerevisiae mitochondrial DNA (mt DNA)

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Page 1: A rapid miniprep method for the preparation of yeast ......Two protocols have been recently proposed to speed up the preparation of Saccharomyces cerevisiae mitochondrial DNA (mt DNA)

A rapid miniprep method for the preparation of yeastmitochondrial DNA

A.Defontaine, F.M.Lecocq and J.N.Hallet*Laboratoire de Cytophysiologie Vegetale, University de Nantes 2, rue de la Houssiniere, 44072Nantes Cedex 03, France

Submitted November 14, 1990

Two protocols have been recently proposed to speed up thepreparation of Saccharomyces cerevisiae mitochondrial DNA (mtDNA). The first (1) is a simplified procedure derived from pre-existing methods using a CsCl gradient, while the other (2) isbased on the isopycnic purification of the mitochondrial fractionfrom lysed spheroplasts. Both the above procedures require anultracentrifugation step. We present here a rapid, simple andinexpensive miniprep method which requires no more than usualbench equipment and which gives reliable results for the routineanalysis of mtDNA from both wild and laboratory strains of 5.cerevisiae.

For each strain, the extraction of mtDNA was performed froman overnight 20 ml culture in YPD (1% yeast extract, 2%peptone, 2% dextrose) at 28°C under shaking. The cells wereharvested by centrifugation at 500 Xg for 5 min and the pellet,which corresponds to 0.3—0.4 g (wet weight) was washed twicein sterile distilled water and then once in 1.2 M sorbitol, 50 mMEDTA, 2% mercaptoethanol. The pellet was then resuspendedin 5 ml of Sol A (0.5 M sorbitol, 10 mM EDTA, 50 mM Tris,pH 7.5) containing 2% mercaptoethanol and 0.2-1 mg/ml ofzymolyase 20 T, and then incubated at 37°C for 45 min undergentle agitation. During this step the majority of the formedspheroplasts were osmotically lysed. The suspension can besonicated using an ultrasonic cell disruptor (Bioruptor UCD 130,Tosho Denki Co Ltd) at 19.3-19.5 KHz for 1 to 5 min (untilthe sample becomes visually more clear). This step, which breaksopen the unlysed protoplasts, was not absolutely necessary butdid improve the final result. The cellular lysate was thentransferred into microfuge tubes and centrifuged at lOOOxg for10 min. The supernatant containing the mitochondria wascentrifuged at 15000xg for 15 min and the crude mitochondria!pellet was carefully washed three or four times with Sol A toeliminate genomic DNA contamination (this step is crucial). Themitochondria were resuspended in 0.2-0.4ml of Sol B (100 mMNaCl, 10 mM EDTA, 1% Sarkosyl, 50 mM Tris, pH 7.8) andallowed to lyse for 30 min at room temperature. The nucleic acidswere then purified from the mitochondrial lysate by extractionwith phenol-chloroform and ethanolic precipitation. RNAs couldbe further degraded by RNAse A. Good yields were obtained(10 to 20 /tg DNA per 0.3-0.4 g of wet weight) and the mtDNA was pure enough (absorption ratio at 260/280 nm =1.8-1.9) for RFLP analysis (fig. 1).

This protocol is efficient for many yeast strains and should takeno more than 4 - 6 hours. It is cheap and easy to perform (sinceit avoids ultracentrifugation steps and the use of carcinogenicdyes) and can be adapted for obtaining much larger amounts ofmtDNA. It is suitable for studying the polymorphism of a largenumber of wild strains or for strain identification.

REFERENCES1 Gargouri.A. (1989) Curr. Genet. 15, 235-237.2. Querol.A. and Barrio.E. (1990) Nucl. Acids Res. 18, 1657.

2 3

Figure 1. Agarose gel electrophoresis of EcoRV digested mtDNA ofSaccharomyces cerevisiae strains. Lanes 2 - 3 : industrial enological strains EG8 and 522 Davis; lanes 4 - 5 wild strains; lanes 7 and 8: undigested and digestedmtDNA of the laboratory strain S 288 C; lane 7, L:L dsRNA; lanes 1 and 6:HindUl restriction fragments of Lambda DNA and HaeUl restriction fragmentsof PhiX DNA.

• To whom correspondence should be addressed