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  Section: Biotechnology Methods » Molecular Biology
 
 
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Plant DNA Extraction

 
     
 
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

A thaliana has a very small haploid genome, and this makes obtaining DNA somewhat difficult. The most notable problem is that DNA is usually contaminated with polysaccharides, which inhibit restriction enzymes, as well as other DNA-modifying enzymes. This problem is most easily solved by using young plants that have not accumulated as much polysaccharide as older plants. The best results are obtained with plants that are 2 to 3 weeks postgerminated.
  1. Harvest plants using forceps—carefully remove any adhering soil by hand.
  2. Grind up the following in a mortar and pestle until no large pieces of tissue remain:
    - 0.5–1.5 g plants
    - 0.5 g of glass beads (75-150 µm) per gram of plants
    - 3 mL proteinase K buffer (0.2 M Tris (pH 8.0), 0.1 M EDTA, 1% Sarkosyl, 100 g/mL proteinase K)
  3. Pour into a 10 mL test tube. Incubate at 45–50°C for 1 hr.
  4. Spin 10 minutes at top speed in table top centrifuge (~3000 rpm)
  5. Decant supernatant to a fresh tube. Adjust volume to 3 mL with proteinase K buffer (with or without proteinase K).
  6. Add 6 mL 100% ethanol at room temperature. Invert to mix.
  7. Spin 10K rpm for 15 min in SS34 rotor. Discard supernatant.
  8. Resuspend pellet in 3 mL Tris-Cl (pH 8.0), 1 mM EDTA (TE). Vortex to resuspend.
  9. Extract with phenol, phenol:chloroform, chloroform.
  10. Add 6 mL 100% ethanol. Invert to mix.
  11. Spin 10K rpm for 15 min in SS34 rotor. Discard supernatant. Air dry pellet briefly.
  12. Resuspend in 4 mL TE. Vortex to resuspend.
  13. Add 4.5 g CsCl, 10 mg/mL ethidium bromide and mix.
  14. Spin 53K rpm 16–20 hrs VTi65 20°C.
This protocol has been optimized for yield at the expense of high molecular weight DNA. The nuclear DNA can be separated from plastid DNA by running the gradients with Hoest dye, rather than ethidium bromide.


Plant DNA Isolation
  1. Prepare 5–20 g of clean, frozen, young leaves taken from plants grown under controlled conditions and exposed to darkness for 2 days prior to isolation. Remove mid-ribs.
  2. Grind leaves in stainless steel blender containing 150–200 mL of ice-cold H buffer at maximum speed for 1 min.
  3. Pour the homogenate in a 250-mL centrifuge bottle (on ice) while filtering through 1 layer of miracloth (Calbiochem) under 4 layers of cheesecloth (all previously wetted with 10 mL clod H buffer).
  4. Centrifuge at 2000 g, 4°C, 20 min.
  5. Discard the green supernatant and resuspend the pellet in 40 mL ice cold HT buffer.
  6. Transfer to a 50-mL teflon tube (Oakridge OK) and centrifuge at 2000 g, 4°C, 10 min. Repeat until pellet of nuclei becomes greyish-white (1 to 3X). If anthocyanins are present in the plant, the pellet will be reddish-brown.
  7. Resuspend the pellet thoroughly in 12 mL of HT buffer, then add 12 mL of lysis buffer.
  8. Immediately, add 23.28 g of powdered CsCl and incubate the tubes at 55°C–60°C for 1 hour with occasional inversion.
  9. After solubilization of the CsCl, centrifuge the tubes at 28000 g, 15°C, 30 min.
  10. Filter the supernatant through 2 layers of cheesecloth into a 38-mL quickseal tube containing 1.47 mL EtBr solution using a 50-mL syringe and a 16-G needle as a funnel. Complete volume with CsCl solution.
  11. DNA is recovered after centrifugation using standard procedures.
    1X TE: 10 mM Tris-HCl (pH 8), 1 mM EDTA
    1X H: 10X H, 400 mL; sucrose, 684 g; β-mercaptoethanol, 8 mL; water to 4000 mL [pH 9.5]
    Lysis buffer: Na-sarcosine, 4 g; Tris base, 2.42 g; Na2EDTA, 2.98 g; water to 200 mL [pH 9.5]
    10X H: spermidine, 20.35 g; spermine, 27.8 g; Na4EDTA 83.24 g; Tris base, 24.2 g; KCl, 119.2 g; water to 1900 mL [pH 9.5]; add Phenyl methyl sulfonyl fluoride (PMSF) solution [7 g in 100 mL of 95% ethanol]
    1X HT: 1X H, 1000 mL; Triton X100, 5 mL
    CsCl solution: CsCl, 97 g; 1X TE, 100 mL
    EtBr solution: EtBr, 1 g; water, 100 mL.

 
     
 
 
     




     
 
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