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実験プロトコール

ゼブラフィッシュ関連

試薬・バッファー類
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).

Bleach 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.

20x PTU
  • 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.

20x Tricaine
  • 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.

抽出・精製関係
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.



細胞培養関係

基本培養プロトコール
リンク先からどうぞ
Cell-based Assay
細胞増殖試験

Protocols