Molecular Biology Problem Solver 30. 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. | is a good idea to consider performing control experiments when using a new lot of polymer for the first time. Structural Uncertainty What is the basic structure of a double-stranded polymer Is it blunt ended Will it have overhangs How long are the overhangs There is no single answer to these questions due to the heterogeneous nature of the product and the impact of the exact conditions used for dissolving the polymer. The buffer composition temperature of dissolution and volume of buffer used will all affect the final structure of the dissolved polymer. Heterogeneous Nature If you add equimolar amounts of a disperse mixture of poly dA and a disperse mixture of poly dT what are the odds that two strands bind perfectly complementary to form a blunt-ended molecule What s the likelihood of generating the same overhang within the entire population of double-stranded molecules Does one strand of poly dA always bind to one strand of poly dT or do multiple strands interact to form concatamers See Figure for examples. Considering the heterogeneous population of the starting material one should assume that a highly heterogeneous population of double-stranded polymers forms. Buffer Composition Double-stranded polynucleotides are usually supplied as lyophilized powders that may or may not contain buffer salts. The pH salt concentration and temperature of the final suspension affect the structure of the dissolved polymer. For example at any specific temperature the strands of poly dA dT resuspended in water dissociate much more frequently than the same polymer dissolved in 100mM sodium chloride. Heating a polymer solution to 85 C for 10 minutes followed by quick chilling on ice produces a different population of polymers compared to poly dA dT dissolved in the same buffer at room temperature. Consider these solution variations when attempting to reproduce your experiments and those cited in the literature. Poly dT Poly dA Poly dT Poly dA Figure Variable products when
