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  Section: Biotechnology Methods » Molecular Biology
 
 
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Southern Blotting (Second Method)

 
     
 
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

Procedure
  1. Electrophoresis of DNA is carried out in a neutral agarose gel system. Prepare a 0.8%–1% agarose gel containing 1X TAE buffer. Ethidium bromide can be added to a final concentration of 0.2 mg/mL.
  2. Apply the samples to the gel.
  3. Run the gel in 1X TAE buffer at 4V/cm until the bromophenol blue indicates that the sample has run for a sufficient distance.
  4. Following electrophoresis, visualize the gel under UV transillumination and photograph it along with a ruler.
  5. (i) Depurination—10 minutes at room temperature with gentle agitation (optional). This step is necessary if target sequences are greater than 10 Kb in size.
    (ii) Denaturation—25 minutes at room temperature with gentle agitation.
    (iii) Neutralization—30 minutes at room temperature with gentle agitation. When using nitrocellulose membranes, the neutralization time should be extended to 45 minutes. Include a rinse in distilled water between each step.
  6. Assemble the capillary blotting apparatus using 10X SSC as the transfer buffer. Allow the DNA to transfer overnight onto Hybond N+.
  7. The following day, disassemble the apparatus, mark the membrane appropriately and fix the DNA to the membrane by UV cross link orbaking (2 hours at 80°;C). For nitrocellulose membranes, bake for 2 hrs, at 80°;C in a vacuum oven.
  1. Hybridization buffer
    - 5X SSC
    - 1 in 20 dilution liquid block (Amersham) or other blocking reagent
    - 0.1% (w/v) SDS
    - 5% (w/v) dextran sulfate
  2. EDTA stock:0.5M EDTA pH8.0
  3. SDS stock:10% or 20% (w/v) SDS
  4. Depurination solution (for Southern blotting)
    - 250 mM HCl
  5. Denaturation solution (for Southern blotting)
    - 1.5M NaCl
    - 0.5M NaOH
  6. Neutralization solution (for Southern blotting)
    - 1.5M NaCl
    - 0.5M Tris-HCl
    - pH adjusted to 7.5
  7. 20X SSC:0.3M Na (3) citrate, 3M NaCl

 
     
 
 
     




     
 
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