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  Section: Biotechnology Methods » Molecular Biology
 
 
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Purification of 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

Materials
  • Spooled DNA
  • SSC buffer
  • Pancreatic ribonuclease A (100 µg/mL)
  • Pronase
  • Sodium lauryl sulfate (SLS)
  • Sodium perchlorate
  • Chloroform:isoamyl alcohol (24:1)
  • 95% and 70% (v/v) ethanol
  • Refrigerated centrifuge, rotor, and tubes
Procedure
  1. Decant off the alcohol, and dissolve the extracted DNA in 30 mL of diluted SSC buffer (0.1 X SSC) in a 125-mL erlenmeyer flask. This will require some time, as polymerized DNA dissolves slowly. Gentle swirling of the material will help.
  2. Add pancreatic ribonuclease A to a final concentration of 100 micrograms/ mL and agitate slowly at 37°C for 1 hour.
  3. Add pronase to a final concentration of 50 micrograms/mL and again agitate slowly at 37°C for another hour.
  4. Add sodium lauryl sulfate (SLS) to make a 1% concentration (w/v) and sodium perchlorate to a final concentration of 1 M. Agitate for 30 minutes at room temperature.
  5. Extract the solution with chloroform:isoamyl alcohol (24:1 v/v) by adding an equal volume and shaking vigorously for at least 15 minutes.
  6. Place the solution into appropriate centrifuge tubes and centrifuge for 5 minutes at 800 xg and 4°C.
  7. Remove the upper aqueous phase, add 2 to 3 volumes of 95% cold ethanol, and respool the DNA from this solution onto a glass rod.
  8. Wash the spooled DNA twice with cold 70% ethanol and store for future analysis.

 
     
 
 
     




     
 
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