TFB1 (RbCl competent-cell buffer 1) recipe
The first buffer of the Hanahan RbCl method for high-efficiency chemically competent E. coli: 30 mM KOAc, 100 mM RbCl, 10 mM CaCl₂, 50 mM MnCl₂, 15% glycerol, pH 5.8.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Potassium acetate | 588 mg | 30 mM |
| RbCl | 2.42 g | 100 mM |
| CaCl₂·2H₂O | 294 mg | 10 mM |
| MnCl₂·4H₂O | 1.98 g | 50 mM |
| Glycerol | 30 mL | 15% |
| Water (distilled) | to final volume |
Steps
- Dissolve in about 80% of the final volume of water.
- Adjust to pH 5.8 with dilute acetic acid.
- Bring to the final volume with water.
- Filter-sterilise through 0.22 µm. (autoclaving precipitates manganese). Store at 4 °C.
Good to know
- Above pH 6 brown MnO₂ precipitates — discard if it turns brown.
- Chill cells grown to OD600 0.4–0.5, resuspend in TFB1 (1/3 culture volume), then in TFB2 (1/25 volume).
FAQ
What if I have no RbCl?
Substituting KCl lowers efficiency a little but is fine for most cloning.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.