DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping
筆記録
– Start the procedure by placing an anesthetized adult fish on a stack of absorbent paper. Now use a sterile razor blade に cut the tail fin and quickly transfer the fish に a tank containing fresh water for recovery. Transfer the tail clip に 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 で an incubator overnight に completely break down the tissue at 55 degrees Celsius with continuous rotation. Next, heat the tube at 95 degrees Celsius for 15 minutes に inactivate proteinase K. Centrifuge the tube に pellet undigested material and transfer the supernatant containing DNA に another tube. Add alcohol に precipitate the DNA and centrifuge again に collect the DNA で 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 に 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 で 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 に 3 millimeters long. Quickly placed the fish で 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 に label both the animal's tank and the tube. Incubate all the collected tissues for at least four hours and up に 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 に three months.
