Exercise 14.1 - Extraction of DNA from Bovine Spleen

LEVEL I

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 over-homogenize 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 4,000 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 two 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 four extractions from above and centrifuge at 20,000 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.

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 (Exercise 14.2).

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