Molecular Biology Problem Solver 54. 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. | increase in the doubling time or a decreased sensitivity to viral infection. This seems to occur when cells are grown in serum-free media after about 30 to 50 passages. At that time a fresh culture is started from frozen cells. IMPLEMENTING THE BACULOVIRUS EXPERIMENT What s the Best Approach to Scale-Up Cells are initially grown in 75 or 150 cm2 flasks and transferred to suspension culture in spinner flasks or shake flasks for scale-up. An advantage of suspension culture is that cells are subjected to less handling so they will attain higher densities than in stationary flasks. The cell volume should not exceed 50 of the flask volume as the oxygen demand increases greatly after infection. There are several types of media that can be used including serum-free preparations and formulations specifically made for T. ni cells. Cells grown in the presence of serum may require a weaning period before being adapted for growth in serum-free media. For the expression of cytoplasmic proteins all types of media will give adequate expression levels but for secreted proteins a low-protein or serum-free preparation may be preferred. An important consideration for secreted proteins is that serumfree media often contains Pluronic a detergent . If a downstream purification step requires a media concentration step pluronic micelles will be concentrated as well and this may affect subsequent chromatography efforts. If large quantities of protein are required it is worth comparing protein expression with a selection of both serum-supplemented and serum-free media preparations as part of an optimization effort. Unfortunately no one media preparation seems to be optimal for all proteins. Many manufacturers of serum-free media occasionally have not been able to meet consumer demand at one time or another so it is worth identifying an alternate commercial source for an acceptable serum-free preparation. Virus stocks should be prepared in serum containing media or serum-supplemented .
