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  Section: Biotechnology Methods » Molecular Biology
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Isolation of Chloroplast DNA

Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  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
  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 a blender with 7 vol LL for maize, and 10 for sorghum. Use MCE at 350 g/L, and if necessary, 5 mL 1 M DIECA/L. Cut leaves into ca. 1" strips prior to blending.
  2. Squeeze through cheesecloth, and Miracloth.
  3. Centrifuge in GSA/JA14 rotors, if necessary. 10 min at 500 rpm GSA;10 min at 570 rpm JA14.
  4. Pellet chloroplasts in GSA/JA14; 15 min at 3000 rpm GSA;15 min at 3100 rpm JA14.
  5. Resuspend with pipette, in LL without MCE, etc. (ca. 300 mL/250 g).
  6. Centrifuge 10 min in SS34/SA600/JA17 rotors: SS34 600 rpm; SA600 530 rpm; JA17 560 rpm.
  7. Pellet chloroplasts 15 min in SS34/SA600/JA17 rotors: SS34 3000 rpm; SA600 2700 rpm; JA17 2800 rpm.
    Use of small rotors is preferable.
  8. Resuspend with pipette in LL, ca. 250 mL/100 g centrifuge, as in step 7.
  9. Resuspend with pipette in LL, ca. 10 mL/100 g if possible.
  10. Add MgCl2 to 10 mM (100 mL 1M/10 mL); DNAse to 100 g/mL (250 mL 2 mg/mL/10 mL). Incubate 60 min at room temperature.
  11. Bring to about 250 mL/100 g with MM; centrifuge as in step 7; resuspend with pipette in MM to ca. 80 mL; centrifuge as in step 7.
  12. Resuspend with pipette in NN; minimum volume, ca. 5 mL/100 g.
  13. Add SDS to 0.5% (250 mL of 10%/5 mL NN). Swirl.
  14. Add Proteinase K to 200 g/mL (500 mL of 20 mg/mL/5 mL). Swirl.
  15. Lyse 60 min at 37°C. (Go to dellaporta for sorghum).
  16. Maize: 3 standard phenol:chloroform extractions, ethanol precipitation.


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