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Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis

  • Ken-Wing Lee
  • Daniel F. BogenhagenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

Abstract

Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA–protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.

Key words

Mitochondria mtDNA nucleoids Mitochondrial RNA 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Pharmacological SciencesStony Brook UniversityStony BrookUSA
  2. 2.Memorial Sloan Kettering Cancer InstituteNew YorkUSA

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