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  Section: Biotechnology Methods » Molecular Biology
 
 
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Extraction of Genomic DNA from Plant Source

 
     
 
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

Principle
The major given protocol describes a rapid method for the isolation of plant DNA without the use of ultracentrifugation. The DNA produced is of moderately high molecular weight, which is suitable for most restriction-end nucleases and genomic blot analysis (Delta portal, et al, 1983). This method also helps to extract DNA from small amounts of tissues and a large number of plant samples, and is very rapid.

Materials
  • Plant tissue
  • Young leaves and callus of hibiscus
  • Pestle and mortar
  • Icebox and ice
  • DNA extraction buffer
  • 5M potassium acetate
  • 3M sodium acetate buffer
  • Phenol choloroform
  • Isoamyl alcohol
  • 100% ethanol (ice cold) 70% ice cold ethanol
  • Sterile eppendorf
  • Microcentrifuge and glass powder
Procedure
  1. Use 500 mg of young plant tissue (stem/leaf/leaf callus of controlled or transformed plant), in a precoated mortar and pestle.
  2. Grind it up inside the icebox with ice, using 0.8 mL of extraction buffer and glass powder.
  3. Add the rest of the buffer. Pour the ground extraction into a test tube.
  4. Add 0.2 mL of 20% SDS, shake, and inoculate at 65°C–75°C for 10 to 15 minutes.
  5. Add 1.5 mL of potassium acetate (5 m).
  6. Shake vigorously, and keep on ice for 20 minutes.

Result
The DNA from plant cells has become extranded and clear bands are seen when the DNA was seen on the gel.

Isolation of DNA From Coconut Endosperm

  • Introduction: The distinctive property of DNA is its behavior upon denaturation The native form of cellular DNA is a helical double-stranded structure. When the native DNA is disrupted, the molecule loses its highly ordered structure and random coils result. Significant molecular dimensions accompany the disruption.
  • Principle: DNA can be isolated from the cells by the phenol extraction method. The cell wall is denatured and chelated by SDS (sodium dodecyl sulfate) or SLS (sodium lauryl sulfate). The tris acts as a buffer. The change in pH of the medium is very low (about 0.3) because of tris. Action of nucleases is minimized by using EDTA. Phenol-chloroform treatment denatures DNA. Isopropanol is necessary for the proper precipitation of DNA, which after being renatured by sodium acetate, is precipitated by cold absolute ethanol.

Materials  
   
Lysis Buffer: 10 mM Tris (pH 8)
  5 mM EDTA
  1% SDS
Phenol  

Chloroform

 

Isopropanol

 

3 M sodium acetate

 

Cold absolute alcohol

 

Sample (coconut endosperm)

 

Procedure

  1. Homogenize 250 mg of coconut endosperm in a mortar and pestle with lysis buffer and make up the volume to 5 mL.
  2. Incubate at 50°C for 5 minutes.
  3. Add 5 mL of 1:1 phenol-chloroform mixture.
  4. Centrifuge for 10 minutes at 3000 rpm.
  5. To the supernatant, add an equal volume of 24:1 chloroform-isopropanol mixture.
  6. To the organic phase, add 120th volume of 3M sodium acetate and 2 volumes of cold absolute alcohol.
  7. A white turbidity is developed.
  8. By stirring it with a glass rod, fibrous DNA strands are collected on the glass rod.
Result
White DNA is observed in the form of fibers. This is then confirmed with the help of electrophoresis. On electrophoresis, it has produced a single band.

 
     
 
 
     




     
 
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