Molecular Biology Problem Solver 42. 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. | Signal Duration Will you need to detect a signal from your blot several times over a period of hours or days Are you pursuing a low-copy target that requires an exposure time of days or weeks Would you prefer a short-lived signal to avoid stripping a blot prior to re-probing Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal eliminating the stripping step prior to re-probing Peterhaensel Obermaier and Rueger 1998 . The effects and implications of stripping are discussed in greater detail later in the chapter. The practical lifetime of common radiolabels is several days to weeks and is dependent on the label the ligand and its environment as discussed in Chapter 6 Working Safely with Radioactive Materials. Some nonradioactive systems based on alkaline phosphatase can generate signals lasting 10 days without marked reduction personal observation . Some chemifluorescent systems generate a fluorescent precipitate capable of producing a cumulative signal much like isotopes. The functional lifetime of fluorescent labels will vary with the chemical nature of the fluorescent tag and the methodology of the application. For example signal duration of a fluorescent tag could be defined by the number of times the chromophore can be excited to produce a fluorescent emission. Some tags can only be excited scanned once or a few times while others are much more stable. Consult the manufacturer of the labeled product for this type of stability information. In systems where an enyzme catalyzes the production of a reagent required for fluorescence the enzyme s half-life and sufficient presence of fresh substrate can limit the duration of the signal. Will the Label Be Efficiently Incorporated into the Probe The effects of label size location and linkage method on the incorporation of nucleotides into DNA or RNA are enzymedependent and can be difficult to predict. Small side chains can inhibit nucleic acid synthesis Racine Zhu
