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  Section: Biotechnology Methods » Molecular Biology
 
 
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Preparation of Genomic DNA from Bacteria

 
     
 
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
  • TE buffer
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 20 mg/mL proteinase K
  • Phenol/chloroform (50:50)
  • Isopropanol
  • 70% ethanol
  • 3M sodium| acetate pH 5.2 phase Lock gel (5 prime, 3 prime)
Procedure
  1. Grow E. coli culture overnight in rich broth. Transfer 1.5 mL to a microcentrifuge tube and spin 2 min. Decant the supernatant. Repeat with another 1.5 mL of cells. Drain well onto a Kimwipe.
  2. Resuspend the pellet in 467 µL TE buffer by repeated pipetting. Add 30 mL of 10% SDS and 3 µL of 20 mg/mL proteinase K, mix, and incubate 1 hr at 37°C.
  3. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
    Caution: Phenol causes severe burns. Wear gloves, goggles, and a lab coat, and keep tubes capped tightly.
    Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin 2 min.
  4. Transfer the upper aqueous layer phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a New Phase Lock GelTM tube and spin 5 minutes. Transfer the upper aqueous phase to a new tube.
  5. Add 1/10 volume of sodium acetate mix.
  6. Add 0.6 volume of isopropanol and mix gently until the DNA gets precipitates.
  7. Spool DNA onto a glass rod (or Pasteur pipette with a heat-sealed end).
  8. Wash DNA by dipping end of rod into 1 mL of 70% ethanol for 30 seconds.
  9. Resuspend DNA in a 100–200 µL TE buffer. Complete resuspension may take several days.
  10. Store DNA at 4°C short term, –20°C or –80°C long term.
  11. After DNA has dissolved, determining the concentration by measuring the absorbance at 260 nm.

 
     
 
 
     




     
 
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