Mammalian DNA Extraction
(authored by Peggy Skinner; modified by Kathy Paris)
Materials:
- testes or ovaries (may be frozen)-cold
- tissue buffer (.15M NaCl plus .01 M EDTA)-cold
- 10% SDS (sodium dodecyl sulfate)-cold
- 2 M NaCl-cold
- 95% ethanol-cold
- razor blade or scalpel
- mortar and pestle-cold
- ice
- centrifuge tube with stopper
- centrifuge
- Pasteur pipettes
Procedure:
- Solution prep for one class:
- Tissue buffer: Prepare a 0.1M EDTA by dissolving .925 grams of EDTA in 25 ml of distilled water. Add .87 grams of NaCl to 10 ml of the 0.1 M EDTA and dilute to 100 ml with distilled water. Keep on ice in bottles unless refrigerated. Approximately 5 ml per student group is needed.
- SDS: (detergent to break cell membranes): 25 grams of SDS (or tergazyme) and add distilled water to 100 ml. Approximately 1-2 ml is needed per group. Chill in refrigerator or on ice.
- 2 M NaCl: 11.7 grams NaCl and add distilled water to 100 ml. Need approximately 5 ml per group. Chill it.
- 95% ethanol: Add 5 ml distilled water to 95 ml 100% ethanol and put into plastic bottles and the freezer one day ahead of the lab. You will need approximately 5 ml per group. Must be kept on ice.
- Procedures for lab:
- Prepare and chill the tissue buffer.
- Slice and mince a small piece (about 4 mm) of dog, cat or rabbit ovaries or testes and place in the cold mortar and pestle.
- Keeping the mortar on ice and add 2-3 drops of SDS and 1 ml. of tissue buffer. Grind the tissue until you see no chunks except some fat that won't grind. It will look like liver ground up in water-brownish red in color.
- Transfer the mixture into a centrifuge tube and add two times the volume of cold 2 M NaCl to the tube (example: if the tube's original volume is about 10 ml, you would add two times that volume or 20 ml of NaCl--you can just eyeball the difference). Stopper and shake the tube for a least two minutes (until your arm gets tired). Note changes in color and viscosity (thickness) of the tube contents.
- Centrifuge the tube for at least 5-7 minutes at top speed. The tube will have a precipitate with a supernatant (floating above the precipitate). The supernatant contains the DNA.
- Decant the aqueous upper layer into another tube (leave the precipitate alone). Using a Pasteur pipette, add chilled 95% ethanol to about 2 times the volume of your DNA extract. Use a glass rod or tip of a Pasteur pipette to spool out the whitish DNA fibers.
- The DNA can be stored for over a year this way or it can be redissolved in .01 M EDTA.
- Explanations:
- Testes and ovaries: The choice of tissue will determine the success of DNA extraction. There must be a high nucleus to cytoplasm ratio to guarantee a high yield of DNA fibers and to prevent excessive protein or lipid interference with the procedure. Liver, for example, is a poor choice because of the presence of glycogen. Accessibility is important, and some mammalian tissues, such as the spleen, are excellent, but not easily obtained. Testes/ovaries offer high concentration of DNA and are easily available through a vet or spay/neuter clinic.
- Tissue buffer: The buffer has a dual purpose. It acts as a stabilizing influence on DNA when Na+ ions neutralize and form a shell around the negative charges associated with the phosphate groups of DNA. The buffer also chelates divalent cations like Mg++ thereby inactivating the DNAase which would break down DNA.
- SDS: This has a detergent-like action that dissolves cell membranes and denatures proteins.
- 2M NaCl: This denatures proteins and histones causing the precipitation of the proteins seen as the white precipitate on the bottom of the centrifuge tube.
- Ethanol: Ethanol causes the precipitation of DNA strands because it excludes hydrophilic molecules.
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