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