TB

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Uses

TB buffer is used to make chemically competent cells by the Inoue method Preparing chemically competent cells (Inoue)

Materials

  • PIPES 0.5M solution pH 6.7
  • Manganese chloride tetrahydrate
  • Calcium chloride dihydrate
  • Potassium chloride
  • Potassium hydroxide 1M solution
  • Sterile 0.22 μm bottle top filter
  • vacuum source
  • clean glassware (no detergent residue -- clean with 70% EtOH)

Preparation

For a 1 liter solution add:

  • 250 mM potassium chloride (18.65 g)
  • 15 mM calcium chloride (2.2 g)
  • 10 mM PIPES (20 ml of an 0.5 M solution)

Adjust pH to 6.7 with KOH.

Add:

  • 15 mM manganese chloride (10.88 g)

Sterile filter with a pre-washed 0.22 μm filter and store indefinitely at 4 degrees.

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