Molecular Biology Problem Solver 49. 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. | cloning to generate the insert. Products should be analyzed by agarose gel electrophoresis to determine if DNA of the predicted size was inserted in the vector. As an alternative PCR can be done using as template a small scraping from a colony on the plate. Amplification of the plasmid DNA contained in the cells using the same primers used in cloning or primers that anneal to flanking vector sequences should show a band of the predicted size. This latter method does not confirm the presence of the restriction sites used in cloning but has the advantage of being rapid. Once the presence of an insert of the correct size is confirmed the DNA sequence at the cloning junctions should be determined. It is not uncommon for a primer sequence to be synthesized with an error whether by faulty design or at the hands of the oligo supplier. DNA sequencing to confirm the cloning junctions should be done in parallel with a small-scale expression experiment in which a 1 to 2 ml culture is grown and induced according to a standard protocol. It is important to include a culture that is transformed with the parent expression vector as a negative control in this screening experiment. Following centrifugation the cell pellet should be suspended in SDS-PAGE loading buffer and a small amount loaded on an SDS-acrylamide gel. The viscosity of the whole cell lysate caused by the release of genomic DNA may make gel loading difficult. However addition of extra 1x loading buffer DNaseI 10mg ml extended heating of the sample or sonication should alleviate the problem. After electrophoresis the gel should be stained . Coomassie Blue to visualize the proteins in the whole cell lysate. If expression is good an induced band will clearly be seen at the predicted molecular weight and this will be absent in the no-insert control culture. If no band is visible and the restriction digestion DNA sequencing data indicate that all is well don t despair. Perform an immunoblot of an SDS-acrylamide gel. .
