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  Section: Biotechnology Methods » Molecular Biology
 
 
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Extraction of DNA from Bovine Spleen

 
     
 
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
  • Bovine spleen
  • Saline citrate buffer (SSC)
  • Chilled blender
  • Refrigerated preparative centrifuge
  • 2.6 M NaCl
  • 95% (v/v) ethanol
Procedure
  1. Weigh out approximately 15 grams of frozen bovine spleen. Record the exact weight for future reference.
    Weight of the liver ___________ gm
  2. Drop the pieces of spleen one at a time into a chilled blender containing 150 mL of cold citrate-saline buffer (SSC). Continue to homogenize the tissue until it is thoroughly macerated. Do not overhomogenize and allow the contents to warm up. Proper procedure should take about 30–60 seconds of blending.
  3. Pour the homogenate into nalgene centrifuge tubes and centrifuge at 4000 xg for 15 minutes at 4°C.
  4. Decant the supernatant and discard. The supernatant contains most of the materials that are soluble in physiological
    buffer. RNA, protein, and many carbohydrates are found in this portion. The pellet contains most of the DNA, but it is complexed and in the form of DNP. The pellet also contains any cell debris and unbroken cells resulting from the homogenization.
  5. Resuspend the pellet in about 20 mL of saline/citrate buffer by gently stirring with a glass rod. If the pellet is packed hard and will not disperse easily, you may use a vortex mixer to aid in the dispersion.
  6. Recentrifuge as in step 3. Discard the supernatant.
  7. Add 20 mL of cold 2.6 M NaCl. Break up the pellet with a glass rod, close the centrifuge tube with a tight-fitting cap and shake vigorously. DNA is soluble in cold NaCl and will also dissociate from the protein. Pour off the liquid portion from this procedure and save for next step. Add another 20 mL of cold 2.6 M NaCl and shake vigorously. Continue to do this for 2 more extractions. It is important that the salt be kept cold (use an ice bath) and that the shaking be vigorous. Breaking the pellet with a glass rod may also help.
  8. Combine all 4 extractions from above and centrifuge at 20000 xg for 20 minutes. This will pellet the insoluble proteins.
  9. Pour the supernatant carefully into a liter beaker and slowly add 2.3 volumes of cold 95% ethanol allowing it to pour down the side of the beaker and layer on top of the aqueous supernatant.
  10. Collect the DNA by gently stirring the mixture together. DNA, if it is highly polymerized, will “spool” onto a clean glass rod as the salt solution is mixed with the alcohol. It can then be removed from the solution in the beaker, washed twice with cold 70% ethanol and placed into 70% ethanol or lyophilized for storage.
Notes
DNA spooled by this procedure is impure. Before it can be used for further analysis, care must be taken to remove contaminating protein, RNA, and carbohydrate. There are a number of means to accomplish this, most involving either enzyme digestions (pronase, amylase, and RNAse) or differential salt solubility or combinations of these techniques.

 
     
 
 
     




     
 
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