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Hortaea werneckii transformation media and buffers
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Hortaea werneckii transformation media and buffers

Introduction

List of solutions and growth media used for the transformation of H. werneckii. Updated on November 11, 2024 by José Vargas-Muñiz

Materials

  • Osmotic Media (1.2 M MgSO4, 10 mM sodium phosphate buffer, stored at 4 ˚C)
    • 500 ml: 149.39 MgSO4-7H20, 5 ml 1 M sodium phosphate buffer pH 6.5; adjust pH to 5.8 with 1 M Na2HPO4. Filter sterilized.
  • Trapping Buffer (0.6 M sorbitol, 0.1 M Tris-HCl, stored at 4 ˚C)
    • 1 L: 109.3 g sorbitol, 100 ml 1 M Tris-HCl, pH 7. Autoclave
  • STC (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl pH 7.5 stored at 4 ˚C)
    • 1 L: 218.6 g sorbitol, 1.5 g CaCl2, 10 ml 1 M Tris-HCl, pH 7.5. Autoclave
  • PEG-CaCl2 (60% PEG, 50 mM CaCl2, 50 mM Tris-HCl pH 7.5 stored at RT)
    • 100 ml: 60 g PEG 3350, 0.15CaCl2, 5 ml 1 M Tris-HCl pH 7.5. Autoclave
  • 20X Salt Solution (Stored at RT)
    • NaNO3 120 g (Sodium nitrate)
    • KCl 10.4 g (Potassium chloride)
    • MgSO4 · 7H20 10.4 g (Magnesium sulfate)
    • KH2PO4 30.4 g (Potassium dihydrogenphosphate)
    • Water up to 1 L
  • Trace Elements Solution (Stored at RT, Shake before use)
    • ZnSO4 · 7H20 2.2 g (Zinc sulfate)
    • H3BO3 1.1 g (Boric acid)
    • MnCl2 · 4H20 0.5 g (Manganese chloride)
    • FeSO4 · 7H20 0.5 g (Ferrous sulfate)
    • CoCl2 · 5H20 0.16 g (Phosgene)
    • CuSO4· 5H20 0.16 g (Cupric sulfate)
    • (NH4)6Mo7O24· 4H20 0.11 g (Ammonium heptamolybdate)
    • Na4EDTA 5.0 g (Tetrasodium ethylenediaminetetraacetate)
    • Add the solids to 80 ml of water, dissolving each completely before adding the next. Heat the solution to boiling, cool to 60°C, adjust the pH to 6.5-6.8 with KOH pellets, cool to room temperature, and adjust the volume to 100 ml with water.
  • Glucose Minimum Media (GMM) Agar (1 L, stored at RT)
    • 20X Salt Solution 50 ml
    • Trace Elements 1 ml
    • D-Glucose (Dextrose) 10 g
    • Agar 15 g (1.5%)
    • pH to 6.5, water to 1 L, autoclave
  • Stabilization Minimum Media (SMM) Agar (1 L, stored at RT)
    • 20X Salt Solution 50 ml
    • Trace Elements 1 ml
    • Glucose 10 g
    • Sorbitol 218.6 g (1.2 M)
    • Yeast Extract 1 g
    • Agar 15 g
    • pH to 6.5, water to 1 L, autoclave
  • SMM Top Agar (0.7% w/v of agar) ( 1L, , stored at RT)
    • 20X Salt Solution 50 ml
    • Trace Elements 1 ml
    • Glucose 10 g
    • Sorbitol 218.6 g (1.2 M)
    • Yeast Extract 1 g
    • Agar 7 g
    • pH to 6.5, water to 1 L, autoclave
  • Cas9 working buffer (20 mM HEPES, 150 mM KCl, pH 7.5) (500 ml, stored RT)
    • HEPES 2.38 g
    • KCl 5.59 g
    • Nuclease-free water (up to 500 ml after pH balancing)

Procedure

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