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  Section: Biotechnology Methods » Molecular Biology
 
 
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Southern Analysis of Mouse Toe/Tail 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
  • Tail buffer 50 mL
  • Phenol/chloroform (1:1 mixture)
  • 0.5M EDTA
  • 4M NH4Ac
  • Absolute EtOH
  • 70% EtOH
  • Tris/EDTA pH 7.5
  • Proteinase K (20 mg/mL dissolved in distilled H2O)


Procedure
  1. This works best with mice that are at weaning age. Take 1 toe or 1 mm of tail and place it in a ependorf tube. Don’t take more than this, it isn’t necessary, and too much material interferes further down the track. Also, if the amounts taken are consistant between samples, then the amounts used for the Southern will be even between samples. If there are a number of samples to be collected, place tubes on ice.
  2. Make up a mix of tail buffer and Proteinase K, allowing 600 mL of tail buffer with 500 mg/mL Proteinase K for each sample (plus an extra dose in case of inaccurate pipetting). Place in a waterbath at 55°;C all day, or at least 2 hrs.
  3. Samples are transferred to hot air shaker overnight at 62°;C at a mediumshake speed.
  4. To each eppendorf, add 500 mL of phenol/chloroform (lower phase of mix). Mix by inversion/shake (don’t vortex as this shears the DNA). Spin at 13000 rpm for 2 minutes. Collect supernatant, being careful to avoid the Interphase.
  5. Place supernatant in a fresh eppendorf tube and add:
    - 2 µL 0.5M EDTA pH 8.0
    - 200 µL 4M NH4Ac
    - 800 µL isopropanol
  6. Mix by inversion/shake, Spin at 13000 rpm for 5 minutes. Remove and discard supernatant, being extremely careful not to disturb the pellet.
  7. To the tube with the pellet, add 200 µL 70% EtOH and vortex. Spin at 13000 rpm for 2 minutes. Remove and discard supernatant (watch the pellet).
  8. Add 200 mL TE and vortex, allowing DNA to resuspend at room temperature.
  9. Once DNA has resuspendend, digests may be set up. 30 mL of DNA/TE suspension per digest should be sufficient, RNAse is not necessary. Digest overnight, precipitate with 12 mL 5 m NaCl, 600 mL EtOH and resuspend pellet in 18 µL TE and 7 µL dye. Allow to dissolve for 20 min RT and heat to 37°;C for 5–10 min before loading.

 
     
 
 
     




     
 
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