Encyclopedia of Experiments: Biology
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Transcript
- Start the procedure by placing an anesthetized adult fish on a stack of absorbent paper. Now use a sterile razor blade to cut the tail fin and quickly transfer the fish to a tank containing fresh water for recovery. Transfer the tail clip to a clean tube containing a lysis buffer. The lysis buffer contains nonionic detergents, which solubilize the plasma membrane and the nuclear membrane of a cell. The buffer also contains an enzyme called proteinase K that breaks down proteins and enables the release of DNA into the lysate. It also degrades nucleases, protecting the DNA from nuclease digestion.
Now, leave the tubes with tail clips in an incubator overnight to completely break down the tissue at 55 degrees Celsius with continuous rotation. Next, heat the tube at 95 degrees Celsius for 15 minutes to inactivate proteinase K. Centrifuge the tube to pellet undigested material and transfer the supernatant containing DNA to another tube. Add alcohol to precipitate the DNA and centrifuge again to collect the DNA in a pellet. In the following protocol, we will isolate DNA from the tail clip of an adult zebrafish and from a whole embryo.
- On the day of the DNA preparation, add fresh proteinase K to the lysis buffer at a concentration of 1 milligram per milliliter. Tissue can be collected from an adult fish using a fin clip or from an embryonic fish.
First, anesthetize the fish in tricaine solution. Wait until the gill movements slow. Then, put the fish on a stack of tissues and, with a sterile razor blade, cut off a small piece of the tail fin about 2 to 3 millimeters long. Quickly placed the fish in a labeled tank with fresh water for recovery.
Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with one hundred microliters of DNA lysis buffer. Be sure to label both the animal's tank and the tube. Incubate all the collected tissues for at least four hours and up to overnight at 55 degrees Celsius.
After the incubation, inactivate the proteinase K by heating the tubes at 95 degrees Celsius for 15 minutes. These samples should be used for PCR immediately, but can also be stored at minus 20 degrees Celsius for up to three months.
