Molecular Biology Problem Solver 23. 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. | then either heating to 100 C for 15 minutes or autoclaving for 15 minutes to eliminate RNase. Electrophoresis apparatus used for RNA analysis can be made RNase-free by filling with a 3 hydrogen peroxide solution incubating for 10 minutes at room temperature and rinsing with DEPC-treated water. When preparing RNase-free solutions wear gloves and change them often. Regardless of the method used to prepare RNase-free solutions keep in mind that they can easily become contaminated after preparation. This occurs when solutions are open and used regularly or when they are shared with others. It is wise to prepare small volumes of solutions and aliquot larger volumes into RNase-free containers. Solutions should be clearly labeled RNase-free to avoid contamination and should only be used with RNase-free pipettes and pipette tips. Also adhere to the maxim when in doubt throw it out. How Does DEPC Inhibit RNase The most common method of preparing RNase-free solutions is diethylpyrocarbonate DEPC treatment. DEPC inactivates RNases by carboxyethylation of specific amino acid side chains in the protein Brown 1991 . DEPC is a suspected carcinogen and it should always be used with the proper precautions. How Are DEPC-Treated Solutions Prepared Is More DEPC Better Most protocols specify adding DEPC to solutions at a concentration followed by mixing and room temperature incubation for several hours to overnight. The container lid should be loosened for the extended incubation because a considerable amount of pressure can form during the reaction. Finally the solution is autoclaved this inactivates the residual DEPC by hydrolysis and releases CO2 and EtOH as by-products. The EtOH by-product can combine with trace carboxylic acid contaminates in the vessel to form volatile esters which impart a slightly fruity smell to the solution. This does not mean that trace DEPC remains in solution. DEPC has a half-life of 30 minutes in water and at a DEPC concentration of solutions .
