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  Section: Biotechnology Methods » Molecular Biology
 
 
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Isolation of Mitochondrial DNA

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method
  1. Grind in mortar and pestle or Waring blender with 5 to 7 volumes buffer per 50 g tissue.. Use MCE at 350 l/L, and if necessary, with 5 mL 1 M DIECA/L.
  2. Squeeze through cheesecloth, 2 layers of Miracloth 10 minutes at 1000 g.
  3. Decant supernatant and centrifuge 10 minutes at 15900 g.
  4. Resuspend each pellet in a few drops of buffer G with paint brush; combine; bring to about 10 mL/50 g, 15 mL/75 g.
  5. 10 min at 1000 g; pour off most; swirl pellet to remove fluffy layer; combine.
  6. Bring supernatant to 10 mM MgCl2 (100 mL 1M/10 mL). Bring to 20 g DNAse/mL (100 mL 2 mg/mL/10 mL) 60 min at 4°C.
  7. Underlay shelf buffer, 20 mL/10–15 mL; always use 20 mL or more, 20 minutes at 12000 g.
  8. Resuspend in small volume shelf buffer with brush; bring to about 10 mL/50 to 100 g, 10 minutes at 15900 g.
  9. Resuspend pellets in NN (lysis) buffer (4–5 mL/50 to 75 g).
  10. Add SDS to 0.5% (250 mL of 10%/5 mL NN). Swirl thoroughly.
  11. Add proteinase K to 100 g/mL (20 mg/mL/5 mL NN). Swirl gently 60 minutes at 37°C.
  12. Add equal volume of 3:1 water-saturated phenol, chloroform-isoamyl alcohol mixture. Emulsify 5 to 10 minutes at 7000 g.
  13. Collect supernatant; repeat 17 and 18: 3 total extractions.
  14. Final supernatant; add 0.1 volume 8 M Ammonium acetate; then add 2 volumes of absolute ethanol.
  15. 60 min, –80°C; 10 min at 8000–9000 g; drain; add equal volume 70% ethanol; let sit 10 min; 10 min at 8000–9000 g; drain dry. Vacuum dry pellet, 30 min. Two small corex tubes are better than one 130-mL Corex. Add 100 to 500 mL 0.1X NTE, 10 mL RNAse mixture. Typically use 500 mL per 50 g tissue.
  16. Hydrate 30 min, 37°C.

A Plant Nuclear DNA Preparation
Procedure
This method can be used to prepare petunias cv. Rose du Ciel nuclear DNA suitable for restriction enzyme analysis and cloning. Chloroplast DNA contamination will vary depending on the genotype. Greater chloroplast DNA contamination results when this method is used with the “Mitchell” line of petunias.
  1. Young leaves (20 g) from 1- to 3-month-old petunia plants are ground at 4°C with a minimal volume of 0.3 M sucrose, 5 mm MgCl2, 50 mM Tris HCl (pH 8) (HB) in a mortar and pestle. The slurry volume is brought to 100 mL with HB and filtered through 2 layers, then 4 layers, of cheesecloth.
  2. The filtrate is centrifuged at 1000 g for 5 min and the pellet resuspended in 100 mL of HB and repelleted at 1000 g for 5 min. This pellet is resuspended in 100 mL of HB containing 2% Triton X-100 incubated at 4°C for 10 min and centrifuged at 1000 g for 5 min. The pellet is washed with 100 mL of BH with Tirton X100, recentrifuged, and the pellet cleaned of excess liquid with a paper towel.
  3. The pellet is resuspended in 16 mL of 30 mM Tris-HCl (pH 8), 10 mM EDTA (RB), and transferred to a 100-mL flask. 2 mL of 10% (w/v) Sarkosyl is added along with 2 mL of 5 mg/mL pronase. The mixture is heated at 60°C for 5 min and incubated with gentle shaking at 37°C for 5–10 hrs. The solution is highly viscous and contains among other things, starch grains and nuclear debris.
  4. After the incubation, measure the total volume of the lysate and bring it to a volume of 20 mL. Add 20 g of CsCl and dissolve completely over a period of about 2 hours at 4°C.
  5. Bring the mixture to room temperature, and add 2 mL ethidium bromide (10 mg/mL). Mix in gently and thoroughly.
  6. Dispense in centrifuge tubes and centrifuge at 35K rpm for 30 hrs at 15°C. Collect the DNA band under long-wave UV light.
  7. Recentrifuge the DNA and, extract the ethidium bromide with Rβ-saturated isoamyl alcohol and dialyze against 5 mM Tris-HCl (pH 8), 0.25 mM EDTA. Store at 4°C.

 
     
 
 
     




     
 
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