Molecular Biology Problem Solver 25. 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. | time as it may provide important information concerning particular substrate DNAs or alternative reaction conditions for a specific application. What Insight Is Provided by a Restriction Enzyme s Quality Control Data Restriction enzymes are isolated from bacterial strains that contain a variety of other enzyme activities required for normal cell function. These additional activities include other nucleases phosphatases and polymerases as well as other DNA binding proteins that may inhibit restriction enzyme activity. In preparations where trace amounts of these activities remain the end-structure of the resulting DNA fragments may be degraded thus inhibiting subsequent ligation. Likewise plasmid substrates may be nicked thus reducing transformation efficiencies. Ideally the restriction enzyme preparation should be purified to homogeneity and free of any detectable activities that might interfere with digestion or inhibit subsequent reactions planned for the resulting DNA fragments. In order to provide researchers with a practical means to conveniently evaluate the suitability of a given restriction enzyme preparation suppliers include a Certificate of Analysis with each product detailing the preparation s performance in a defined set of Quality Control Assays. In order to establish a standard reference for the amount of enzyme and substrate used in these assays each supplier must first define the unit substrate and reaction conditions for each product. Unit Definition A unit of restriction endonuclease is defined as the amount of enzyme required to completely cleave 1 mg of substrate DNA suspended in 50 ml of the recommended reaction buffer in one hour at the recommended assay buffer and temperature. The DNA most often used is bacteriophage Lambda or another well-characterized substrate. Note that the unit definition is not based on classic enzyme kinetics. The enzyme molar concentration is in excess. A complete digest is determined by the visualized pattern of .
