TB

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

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 &mu;m 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 &mu;m filter and store indefinitely at 4 degrees.