by high-performance chromatography, but when such equipment is unavailable, thin layer chromatography can provide qualitative data(Table 10.3) How Should You Prepare, Quantitate, and Adjust the ph of Small and Large Volumes of nucleotides? o The following procedure can be used to prepare solutions of oxynucleotides, ribonucleotides, and dideoxynucleotides pr vided that the different formula weights are taken into account A 100 mM solution of a solid nucleotide triphosphate is pre pared by dissolving about 60mg per ml in purified H,O The exact weight will depend on the formula weight, which will vary by nucleotide, supplier, and salt form. As solid nucleotide triphos phates are very unstable at room temperature, they should be stored frozen until immediately before preparing a solution pectroscopy The most accurate method of quantifying a solution is to measure the absorbance by UV spectrophotometry. a dilution should be made to obtain a sample within the linear range of the spectrophotometer. The sample should be analyzed at the specifi Amax for the nucleotide being used. The concentration can then be obtained by multiplying the UV absorbance reading by the dilution factor, and dividing by the characteristic Am for that nucleotide. These data are provided in Table 10.2 Table 0.3 TLc conditions to monitor dntP Degradation RA Principal R, Trac 0.35(dADP) dcTP 0.21(dCDP) dGTP .34(dGDP) B dTTP 0.14 . 21(dTDP) Note: Solvent System A: Isobutyric acid/concentrated NH,OH/water, 66/1/33; PH 3.7. Add 10ml of concentrated NhOH to 329 ml of water and mix with 661 ml of isobu. uric acid. Solvent System B: Isobutyric acid/concentrated NH OHA water, 57/4/39: pH 43. Add 38ml of concentrated NH,OH o 385 ml of water and mix with 577 ml of isobutyric acid. TLC Plates: Eastman chromagram sheets (#13181 silica gel and #13254 cellulose). Nucleotides, Oligonucle and Polynucleotides 273by high-performance chromatography, but when such equipment is unavailable, thin layer chromatography can provide qualitative data (Table 10.3). How Should You Prepare, Quantitate, and Adjust the pH of Small and Large Volumes of Nucleotides? The following procedure can be used to prepare solutions of deoxynucleotides, ribonucleotides, and dideoxynucleotides pro￾vided that the different formula weights are taken into account. A 100 mM solution of a solid nucleotide triphosphate is pre￾pared by dissolving about 60mg per ml in purified H2O. The exact weight will depend on the formula weight, which will vary by nucleotide, supplier, and salt form. As solid nucleotide triphos￾phates are very unstable at room temperature, they should be stored frozen until immediately before preparing a solution. Quantitation Spectroscopy The most accurate method of quantifying a solution is to measure the absorbance by UV spectrophotometry. A dilution should be made to obtain a sample within the linear range of the spectrophotometer. The sample should be analyzed at the specific lmax for the nucleotide being used. The concentration can then be obtained by multiplying the UV absorbance reading by the dilution factor, and dividing by the characteristic Am for that nucleotide. These data are provided in Table 10.2. Nucleotides, Oligonucleotides, and Polynucleotides 273 Table 10.3 TLC Conditions to Monitor dNTP Degradation Solvent dNTP Rf, Principal Rf, Trace System dATP 0.25 0.35 (dADP) A dCTP 0.15 0.21 (dCDP) A dGTP 0.27 0.34 (dGDP) B dTTP 0.14 0.21 (dTDP) A Note: Solvent System A: Isobutyric acid/concentrated NH4OH/water, 66/1/33; pH 3.7. Add 10 ml of concentrated NH4OH to 329 ml of water and mix with 661 ml of isobu￾tyric acid. Solvent System B: Isobutyric acid/concentrated NH4OH/ water, 57/4/39; pH 4.3. Add 38 ml of concentrated NH4OH to 385 ml of water and mix with 577 ml of isobutyric acid. TLC Plates: Eastman chromagram sheets (#13181 silica gel and #13254 cellulose)