Molecular Biology Problem Solver 44. 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. | require longer hybridization times than single-stranded probes end-labeled oligonucleotide because reassociation of doublestranded probes in solution competes with annealing events of probes to target. At 50 to 75 reassociation free probe concentration has dwindled to amounts that make further incubation futile. Hybridization time for a double-stranded probe can therefore be deduced from its reassociation rate Anderson 1999 . Glimartin 1996 discusses methods to predict hybridization times for single-stranded probes as does Anderson 1999 . Other variables of hybridization time include probe length and complexity probe concentration reaction volume and buffer concentration. Buffer formulations containing higher concentrations 10ng ml of probe and or rate accelerators or blots with high target concentrations may require as little as 1 hour for hybridization. Prolonged hybridization in systems of increased hybridization rate will lead to background problems. The shortest possible hybridization time can be tested for by dot blot analysis. Standard buffers usually require hybridization times between 6 and 24 hours. Plateauing of signal sets the upper limit for hybridization time. Again optimization of hybridization time by a series of dot blot experiments removed and washed at different times is recommended. Plaque or colony lifts may benefit from extended hybridization times if large numbers of filters are simultaneously hybridized. What Are the Functions of the Components of a Typical Hybridization Buffer Hybridization buffers could be classified as one of two types denaturing buffers which lower the melting temperatures and thus hybridization temperatures of nucleic acid hybrids . formamide buffers and salt detergent based buffers which require higher hybridization temperatures such as sodium phosphate buffer as per Church and Gilbert 1984 . Denaturants Denaturing buffers are preferred if membrane probe or label are known to be less stable at elevated temperatures.
