dNTP Stock Solutions
pH-adjusted solutions of dNTPs are available from many commercial manufacturers; however, it is also possible to prepare dNTP stocks from powdered sodium salts. Stock solutions usually contain a single dNTP at a concentration of 10 or 20 mm, according to the needs of the investigator. These stocks are stored at −20°C in small aliquots, and they may be diluted to generate, for example, solutions that contain all four dNTPs at a concentration of 5 mm.
A microbalance is used to weigh out the required amounts of dNTP into sterile microcentrifuge tubes. Either use a disposable spatula or clean the spatula well with ethanol between each weighing when making up solutions of different dNTPs. The table below shows the amount of solid, anhydrous deoxynucleotide required to make 1 mL of a 20 mm stock solution.
| Deoxynucleotide | F.W. | Amount (in milligrams) required to make 1 mL of a 20 mm solution |
|---|---|---|
| dATP | 491.2 | 9.82 |
| dCTP | 467.2 | 9.34 |
| dTTP | 482.2 | 9.64 |
| dGTP | 507.2 | 10.14 |
| Dissolve the deoxynucleotides in a small volume of H2O and then use pH paper and an automatic pipetting device to adjust the pH to ∼8.0 by adding small amounts of 2 n NaOH until the pH reaches 8.0. Alternatively, the stock solutions may be generated from monosodium salts of the deoxynucleotide triphosphates by dissolving an appropriate amount of the solid in 1.0 mL of H2O. Mix the contents of the tube well and store the stock solution in small aliquots at −20°C. | ||
