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Team:EPF-Lausanne/Protocols/Competent cells. Protocol II
From 2011.igem.org
Preparing chemically 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.
