DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

프로토콜

1. DNA Preparation

1. Prepare DNA lysis buffer: 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.3% Tween 20, 0.3% NP40. Add fresh Proteinase K to a final concentration of 1 mg/ml on the day of use.
2. Tissue collection:
   1. For adult fish fin clip:
      1. Anesthetize fish: Place the fish in 0.004% MS-222 (tricaine) solution. Wait until gill movement slows.
      2. Put fish on a stack of 5-10 Kimwipes and cut a small piece of the tail fin, about 2-3 mm, with a sterile razor blade.
      3. Quickly place the fish in a labeled tank with fresh water for recovery; carefully label both the tank and corresponding tube that will contain the fin clip. Change the water and feed the fish every other day.
      4. Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with 100 μl DNA lysis buffer.
      5. Discard the pipette tip, and discard the razor blade into a sharps container.
   2. For embryo tail clip:
      1. Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.
      2. Cut a piece of tail with forceps under a dissecting microscope.
      3. Put the tail into a labeled tube filled with 30 μl DNA lysis buffer.
      4. Quickly put the embryo into a tube containing 100 μl 4% paraformaldehyde.
      5. Label tubes with embryos and their corresponding tail tubes.
      6. Store the tubes with embryos at 4 °C overnight for fixation.
      7. Clean the forceps using Milli-Q water and Kimwipes between each embryo to minimize contamination.
   3. For whole embryos:
      1. Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.
      2. Put 1-5 embryos in a tube filled with 100 μl DNA lysis buffer. Dechorionation is not necessary.
3. DNA digestion
   Incubate tubes with tissue (fin or tail clips or embryos) at 55 °C for 4 hr up to overnight.
4. Inactivate the Proteinase K
   Heat tubes to 95 °C for 15 min. DNA should be used for PCR immediately, or stored at -20 °C for up to 3 months.

자료

Name	Company	Catalog Number	Comments
100 Reaction LightScanner Master Mix	BioFire	HRLS-ASY-0002	www.biofiredx.com
Tricaine
Paraformaldehyde