2X HBS (for calcium-phosphate transfection, pH 7.05) recipe
The buffer mixed with DNA–CaCl₂ in calcium-phosphate transfection to form a fine precipitate: 50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄, pH 7.05.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| HEPES (free acid) | 1.192 g | 50 mM |
| NaCl | 1.636 g | 280 mM |
| Na₂HPO₄ (anhydrous) | 21.3 mg | 1.5 mM |
| Water (distilled) | to final volume |
Steps
- Dissolve in about 80% of the final volume of water.
- Adjust precisely to pH 7.05 with NaOH (±0.05 changes efficiency markedly).
- Bring to the final volume with water.
- Filter-sterilise through 0.22 µm. Aliquot and store at −20 °C.
Good to know
- Add DNA + water + 2.5 M CaCl₂ (250 mM final) dropwise to an equal volume of 2X HBS, wait 20 min, then add to cells.
- Many labs make batches at pH 6.95, 7.05 and 7.12 and keep the best one.
FAQ
The precipitate is too coarse.
The pH is probably too high or it stood too long; you want a fine, hazy precipitate.
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.