Buffer B70 (RNase-free)

5.6 mL potassium acetate (KAc; 5 M)

400 μL sodium fluoride (Na F; 1 M)

16 mL β-glycerophosphate (0.5 M; e.g., Sigma G6251)

2 mL magnesium acetate (MgOAc; 1 M)

4 mL Triton X-100 (10%)

80 mL glycerol (50 %)

• 1. To prepare 400 mL, combine the above ingredients.

• 2. Bring the volume to 380 mL with H₂O and add:

  • 380 μL DEPC

• 3. Stir 1 h with a stir bar (shaking causes too much foam) and autoclave the solution.

• 4. Add 20 mL DEPC-treated HEPES-KOH (1M, pH 7.4) and filter-sterilize the solution.

• 5. Where indicated, add the following protease inhibitors immediately before use:

  • 1 mM DTT

  • 1 mM PMSF

  • 2 mM benzamidine

  • 1 μg/mL leupeptin

  • 1 μg/mL pepstatin A

  • 0.1 mg/mL Pefabloc

  • 1X Complete Protease Inhibitor Cocktail Tablets with EDTA (Roche 11697498001)

    When preparing the protease inhibitor stock solutions, use an ethanol-dipped and flamed metal spatula for weighing the individual components. Dissolve components in DEPC-treated H₂O or RNase-free ethanol.