modified by Sue Black
|Testes or thymus glands: frozen or fresh
||2M NaCl - Cold
|Crushed ice in container
||95% Ethanol - Cold
|Tissue buffer - cold
||Mortar and Pestle, on ice
|SDS at room temperature and dropper
|Glass stirring rod
||10 ml Graduated Cylinder
||Stopper for Centrifuge Tubes
- Obtain a piece (approx. 2 cm3) of dog testes, and place it in a mortar which is frozen in ice. Place the mortar in a plastic dish from soft margarine that is 2/3 full of water and freeze the day before.
- Add 4 ml of tissue buffer
(cold) and one squirt of SDS to the mortar containing the testes tissue, and mince the tissue with a razor blade. Then grind (homogenize) the tissue
with the pestle. Always keep the mortar and pestle on ice.
- Pick out the unhomogenized gristle and transfer the entire slurry into a test tube
with stopper and add two times the volume of cold 2M NaCl. (2/3 NaCl sol. and 1/3 slurry).
- Stopper and shake the tube for at least two minutes (your arm should get tired!). Notice the increase in viscosity. Remove the stopper. If the slurry is not in a centrifuge tube, transfer it to one now.
- Centrifuge the tube for 7 minutes at top speed. REMEMBER TO BALANCE THE CENTRIFUGE BY RUNNING YOUR TUBE OPPOSITE SOMEBODY ELSE'S.
A full centrifuge saves everyone time.
- After centrifuging, there will be two layers, a bottom precipitate or pellet of protein, and an upper
liquid supernatant containing the DNA.
- Slowly pour the aqueous upper layer
(supernate) containing the DNA into a test tube and then insert your glass spooling rod.
- Hold the test tube containing the supernate at a 45o angle and add very cold 95% ethanol (let it run gently down the sides of the test tube) to about 2 times the volume of your DNA extract.
- Now turn your spooling rod and watch the whitish-clear DNA fibers come on to the rod. Don't touch the end of the spooling rod with your fingers. The translucent threads of DNA will begin to adhere to the rod at the interface between the two fluids.
Explanation For Materials Or Procedures Used In Extraction
- Testes or thymus gland (sweet bread) provide a high nucleus to cytoplasm ratio which
guarantees a high yield of DNA, and prevents excessive protein or lipid interference
with the procedure. Accessibility is important too. They are available from your
local veterinarian or your local butcher. Tissue must be VERY fresh, not more than
2 days old under refrigeration. It is best to keep it frozen if it is to be used
later. Grind the tissue fresh or frozen.
- Tissue Buffer:
- The buffer has a dual purpose. It acts as a stabilizing influence on DNA by forming
an ionic shell. It also binds to a coenzyme for DNAse (an enzyme that digests DNA
and ruins our long threads) making it inoperative so that it won't destroy the DNA
fibers. EDTA (Ethylene Diamine Tetraacetic Acid) binds divalent cations which are cofactors to DNAse and other DNA cutting enzymes.
- Sodium Dodecyl Sulfate or Sodium Lauryl Sulfate. This detergent-like substance dissolves
lipid cell membranes and denatures proteins. This will allow our DNA to come out
of the cells and into the solution.
- 2M NaCl:
- A strong salt solution that denatures and precipitates proteins (histones are proteins
that DNA wraps around in eukaryotic cells) and therefore exposes DNA.
- Causes the precipitation of DNA so that it can be "spooled," because it excludes
hydrophilic molecules. DNA cannot dissolve in alcohol.
- Why Must Solutions Be Kept Cold?
- Cold will slow the action of any DNAses or proteases (enzymes) which are sometimes
released during the extraction and could ruin your DNA fibers. For the same reason
(enzyme destruction), do not touch the glass rood you use to spool the DNA (There
is allot of DNAse on your fingers).
- Tissue buffer: 0.9 g NaCl + 10 mls of 0.1 M EDTA, diluted to 100 mls with dH2O.
(Stock 0.1 M EDTA: add dH2O to 7.3 g of EDTA until you have a vol. of 250 ml)
EDTA can be purchased very inexpensively through any biological supply house.
SDS: 15% sol. (Do not refrigerate) 15 g in 100 ml dH2O
2M NaCl: add dH2O to 58 g of NaCl until you have a volume of 500 ml.
- Biological Science: Interaction of Experiments and Ideas. 2nd Edition BSCS 1970. (Lab # 15 pgs. 157-159).
Peggy Skinner: The Bush School in Seattle WA. with the advise of Dr. Charles Laird, U. of Washington Zoology Dept.
Sue Black: AP Biology Menlo-Atherton High School, Calif.
Steve Clark: Monterey High School, Calif.
David Crissman: Valencia High School, Valencia, Calif.