TB

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (11:04, 4 February 2009) (view source)
 
(4 intermediate revisions not shown.)
Line 3: Line 3:
==Materials==
==Materials==
-
* PIPES 0.5M solution pH 6.7
+
* PIPES buffer 0.5M solution pH 6.7 (sterilized, made with milli-Q pure H2O, store at -20C)
* Manganese chloride tetrahydrate
* Manganese chloride tetrahydrate
* Calcium chloride dihydrate
* Calcium chloride dihydrate
Line 21: Line 21:
Dissolve  
Dissolve  
-
* 15 mM manganese chloride (10.88 g)
+
* 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.
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.
Line 27: Line 27:
Sterile filter with a pre-washed 0.22 μm filter and store indefinitely at 4 degrees.
Sterile filter with a pre-washed 0.22 μm filter and store indefinitely at 4 degrees.
 +
 +
[[Category:Material]][[Category:Buffers]]

Current revision

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 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.

Personal tools