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

Weigh out approximately 15 grams of frozen bovine spleen. Record the exact weight for future reference.
Weight of the liver ___________ gm
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.
Pour the homogenate into nalgene centrifuge tubes and centrifuge at 4000 xg for 15 minutes at 4°C.
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.
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.
Recentrifuge as in step 3. Discard the supernatant.
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.
Combine all 4 extractions from above and centrifuge at 20000 xg for 20 minutes. This will pellet the insoluble proteins.
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.
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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