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TB buffer for competent cells (Inoue)

Uses

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

Materials

• PIPES buffer 0.5M solution pH 6.7 (sterilized, made with milli-Q pure H2O, store at -20C)
• 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)

to 800 ml of DI water. Adjust pH to 6.7 with 1M KOH.

Dissolve

• 55 mM manganese chloride (10.88 g)

in 100 ml of DI water, add gradually to the solution of the remaining components. Bring solution to 1 liter. The pH of the solution will fall, which is expected.

• Do not attempt to adjust the pH. Adding base after adding manganese will precipitate a yellow/brown hydroxide.

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