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Molecular Biology Problem Solver 29

Molecular Biology Problem Solver 29. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | 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 The following procedure can be used to prepare solutions of deoxynucleotides ribonucleotides and dideoxynucleotides provided that the different formula weights are taken into account. A 100 mM solution of a solid nucleotide triphosphate is prepared 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 triphosphates 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. Table 10.3 TLC Conditions to Monitor dNTP Degradation dNTP Rf Principal Rf Trace Solvent 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 isobutyric 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 . Nucleotides Oligonucleotides and Polynucleotides 273 Weighing One would think that the mass of an extremely