The solution was developed by Dryl (Dryl SJ. 1959. Antigenic transformation in Paramecium aurelia after treatment during autogamy and conjugation. J Protozool 6: S25).
An alternative more in line with modern molecular biology is 10 mM Tris-HCl pH 7.5. But I havent done a proper comparison.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| sterile ddH2O | stock room | ||
| 1M sodium citrate | stock room | ||
| 1M CaCl2 | stock room | ||
| Na2HPO4 | S7907-100G | Merck | SigmaAldrich |
| NaH2PO4 | S0751-100G | Merck | SigmaAldrich |
Begin by making the following stock solutions:
1 M CaCl2
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| CaCl2 | 5.55 g | 1 M |
| TOTAL: | 50 mL |
0.1 M Na2HPO4
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| Na2HPO4 | 709.8 mg | 0.1 M |
| TOTAL: | 50 mL |
0.1 M NaH2PO4
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| NaH2PO4 | 599.9 mg | 0.1 M |
| TOTAL: | 50 mL |
Add reagents in the order listed.
Add CaCl2 last.
This is to avoid precipitation of calcium phosphate.
To avoid precipitation of the CA phosphate, the CaCl2 solution is autoclaved separately from the mixture of sodium salts, and the two solutions are mixed aseptically after cooling.
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 900 mL | |
| Sodium citrate (1 M) | 2 mL | 2 mM |
| NaH2PO4 (0.1 M) | 10 mL | 1 mM |
| Na2HPO4 (0.1 M) | 10 mL | 1 mM |
| CaCl2 (1 M) | 1.5 mL | 1.5 mM |
| sterile ddH2O | 76.5 mL | |
| TOTAL: | 1 Liter |
Autoclaved buffer should be stored in 4°C.