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  Section: Biotechnology Methods » Molecular Biology
 
 
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Southern Blot

 
     
 
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
  1. Run the gel as normal. Often for genomic Southerns, it is desirable to run long gels (18 cm) over 4–6 hrs.
  2. Photograph the gel along with a ruler adjacent to the molecular weight markers as a reference.
  3. Alkali transfer buffer = 0.5M NaOH (20 g/L), 1.5M NaCl (87.66 g/L). Prepare 1 liter for 1 gel and another 750 mL for each additional gel. Beware that this buffer is very dangerous, capable of causing severe eye damage. Use the large volumes involved in this procedure with care and wear protective glasses.
  4. Add the gel to 250 mL alkali transfer buffer, plus 125 mL for each additional gel.
  5. Place gel on rocker with buffer solution for 20 mins. Note that lowpercentage agarose gels must be agitated slowly to prevent tearing.
  6. Keep the remaining 500 mL for the transfer tank.
  7. Wear gloves for the following steps.
  8. Cut 2 pieces of large 3-mm paper (wicks) and 2 pieces about 2-mm smaller than the gel on each edge and 1 piece of nylon (Hybond N+, Amersham) the same size as the gel.
  9. Cut a stack of paper toweling about 2-mm smaller than the gel. The stack needs to be about 6-cm thick.
  10. Prewet nylon in Double Distilled Water (DDW), then soak in alkali transfer buffer.
  11. Add buffer to transfer tank to the level of the platform. Wet the long wicks in transfer buffer and place in the tank.
  12. Place gel on platform and spoon on transfer buffer. Add the nylon and smooth out any bubbles with the back of your finger.
  13. Add the 2 slightly smaller 3-mm filters, the first prewetted, the second dry.
  14. Add the stack of paper towels. Note it is very important that the edges of the towel do not touch the wicks that the gel is sitting on or the transfer will be “shortcircuited”.
  15. Top the stack with a glass or plastic plate and a weight (a bottle with about 200–300 mL H2O is ideal). Too much weight compresses the gel and terminates the transfer early.
  16. Allow to transfer overnight and then remove the stack carefully. Mark the position of the wells on the filter with a biro (and note position of well #1) before removing the filter. Soak the filter in 0.5M Tris pH 7.5, 1.5M NaCl for 5 min after removal from the gel.
  17. Add filter to 200 mL 2X SSC and allow the soak without agitation for 5 min.
  18. Remove filter and blot dry and bake at 80 for 2 hrs or place in autoclave for 10 min when not operating but warm.
  19. Prehybridge with 10 mL Aqua. hybridge (reagents), 1% SDS and 100 mg/mL boiled herring sperm DNA for 20–30 minutes (cloned DNA southern) to several hrs (genomic southern).
  20. Boil probe for 3 min and add to 3 mL of fresh hybridge solution/SDS/ herring DNA. Squeeze prehybridge from the bag and add probe. Seal, avoiding air bubbles, and distribute the probe well. Incubate for 4–6 hrs (cloned DNA) to 16–20 hrs (genomic Southern). Washes are performed in 2 to 0.2X SSC, 0.1% SDS depending on the homology of the probe to target.

 
     
 
 
     




     
 
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