|Zebrafish Breeding Mannual (Japanese only)
30x Danieau's solution
- NaCl 101.7 g (1740 mM), KCl 1.56 g (21 mM), MgSO4•7H2O 2.96 g (12 mM), Ca(NO3)2 4.25 g (18 mM), HEPES buffer 35.75 g (150 mM)
- Dissolved in 1L dH2O and adjust pH to 7.4.
- Autoclaved and store at 4°C.
- Use 100-time dilution (0.3x Danieau's solution).
- 0.1 mL of 5% sodium hypochlorite in 170 ml of 0.3x Danieau's solution.
- Immerse embryos for 5-10 min and wash with 0.3x Danieau's solution more than 3-time.
- N-Phenylthiourea, purchased from Sigma-Aldrich, MW = 152.
- Prepare final 75 microM PTU in 0.3x Danieau's solution.
- For stock, 75 x 20 = 1,500 microM = 1.5 mM
- 1 M = 152g/L, so 1.5 mM = 152x1.5 = 228 mg/L = 22.8 x 4 mg/400mL
- Take 85 mg powder and dissolved in 400mL dH2O, and mixing for O/N.
- Make 50 mL aliquots and store at –30 degree.
- 400 mg tricaine powder (ethyl-m-aminobenzoate methanesulfonate SIGMA A-5040)
97.7 ml DD water, 2.1 ml 1M Tris (pH 9). Adjust to pH 7.
- Store this stock solution in the freezer, in aliquots of 4.2 ml.
- To use tricaine as an anesthetic dilute the 4.2 ml tricaine stock solution
in 100 ml 0.3x Danieau's solution and leave at room temperature.
|Extraction of genomic DNA from zebrafish
- Zebrafish are incubated in 50 μl of lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.2% Triton X-100, 200 μg/ml proteinase K) at 55 °C overnight, followed by incubation at 99 °C for 10 min. The solutions are then kept at 4 °C and used for PCR as the template.