- Bovine spleen
- Saline citrate buffer (SSC)
- Chilled blender
- Refrigerated preparative centrifuge
- 2.6 M NaCl
- 95% (v/v) ethanol
- 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
- 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.
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.