In the following lab you will extract, and partially purify, DNA from animal tissue. DNA has some rather unusual properties such as its long, thin molecular shape. Imagine, for example, a piece of spaghetti long enough to wrap around the world. This is the amount of DNA in a single human cell, if the DNA strand was enlarged to the diameter of spaghetti. But there are certain enzymes which can readily cut it up into many small fragments - and your skin is full of these enzymes. Thus glassware must be carefully cleaned and any surface that will be in contact with DNA must not be touched.
     The four key skills required to get "high molecular weight DNA" is to: work FAST, work COLD, work CLEAN, and DON'T AGITATE the contents. With these guidelines in mind, and by carefully following the procedures described, DNA can be obtained from many types of animal tissues.

PROCEDURE

     In this portion of the lab you will extract DNA from thymus glands (muscle or glandular tissue can be substituted) of an animal. The procedure for this extraction is broken down into 4 parts as described below.

PART A : Isolation of the Cell Nuclei

1. Obtain a chilled mortar and pestle. Keep these chilled by working on ice.

2. Slice or mince a small piece of animal tissue (approximately the size of a pencil      eraser) into the mortar and pestle.

3. Begin to grind the tissue. Continue to grind for approximately 1 to 2 minutes.

     This step of the lab will physically break open many of the cells. Variations on this step include using a kitchen blender in place of the mortar and pestle, and/or adding a sucrose solution while grinding the meat.

PART B :Breaking Open the Nuclei

4. Add 1 ml. of ice cold Homogenization buffer (contains EDTA) and 2 or 3 drops of      SDS solution to the ground meat in the mortar and pestle.

5. Continue to grind the tissue for 1 to 3 minutes, until a "slurry" forms in the bottom of      the mortar and pestle.

     This step of the lab will chemically break open many of the nuclei. Each chemical plays a different role in this process. EDTA, for example, is a common food additive that can remove magnesium and calcium from many biologically important molecules. This has the effect of weakening all membranes in the cell, and inactivating all enzymes that can break down DNA.
     The SDS is a detergent, and can thus dissolve lipids such as grease. This also weakens all membranes in the cell since lipids are a major component of membranes.

PART C: Separating Protein from DNA

6. Transfer the slurry into a cold centrifuge tube.

7. To the volume of slurry in the tube add twice that volume of cold 2 M NaCl. (Another      variation of this lab uses chloroform in an amount equal to the volume of the slurry,
      in place of the NaCl.)

8. Stopper the tube and shake vigorously for at least 2 minutes.

9. Centrifuge the tube at least 5 to 7 minutes at top speed in a clinical centrifuge. A      precipitate will form at the bottom of the test tube, with a liquid on top. (If chloroform      is used, the tube will contain 3 layers of liquids with the DNA in the top layer.)

     This step of the lab will dissolve any protein that was chemically joined to the DNA. Since protein is heavy it can now be separated from the DNA by centrifuging the contents of the test tube.

PART D : Precipitation and Extraction of DNA

10. Pour the liquid portion of the test tube into a larger chilled test tube. (Don't fill the      test tube more than 1/2 full.)

11. Slowly add two volumes of ice cold ethanol to the liquid in the test tube. Pour the      ethanol down the side of the test tube, or down a clean glass rod. Be very careful      not to shake the tube or pour the ethanol too rapidly. If done properly a thin layer of      alcohol should form above the other liquid in the test tube. The DNA should      precipitate out of the alcohol and will appear as a filmy cloud of fibers near the      boundary between the two liquids.

12. Using a long, very clean glass rod begin a slow circular motion where the DNA is      forming. If done properly the DNA will wrap around the glass rod like spaghetti      around a fork. Once removed from the liquid, the DNA often looks glassy and a little      "off-white".

     This part of the lab removed the DNA from the liquid using the alcohol precipitation method. This procedure takes advantage of the low density of alcohol. When poured properly the alcohol will float on top of most other liquids. Now the DNA can be isolated in a thin layer since it will not dissolve in cold ethanol, and therefore precipitates out of the ethanol. Any protein present can dissolve in ethanol, and thus will not precipitate out. The results is relatively pure strands of DNA. Since DNA is sticky it can now be removed using a glass rod and studied in detail.