Exercise 14.1 - Extraction of DNA from Bovine Spleen
- 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 over-homogenize and allow the contents to warm
up. Proper procedure should take about 30-60 seconds of
- Pour the homogenate into nalgene centrifuge tubes and
centrifuge at 4,000 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
- 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
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.
- Combine all four extractions from above and centrifuge at
20,000 xg for 20 minutes. This will pellet the insoluble
- 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
- 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
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
to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com