Molecular Biology Problem Solver 32. 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. | Table Optimizing MgCl2 Concentration for PCR Final Per Component Concentration Reaction 1 mM 2mM 3 mM 4 mM 6 mM 8 mM 10 mM 10x PCR buffer 1 10ml 50 mM forward primer mM 1 ml 50 mM reverse primer mM 1 ml Template DNA Optimum 10ml 100mM MgCl2 Various Various 40 25 mM dNTP mix ml Taq polymerase 5U ml ml H2O To 100 ul To 400ml To 400 ml To 400 ml To 400ml To 400 ml To 400 ml To 400 ml especially important to adjust the primer concentration when the target sequence is rare or the template amount is low. Less primer is needed in these cases too much primer will generate primerdimers or smearing of the product visualized by agarose gel electrophoresis. For most applications it is practical to apply the standard concentrations cited above and to focus effort on optimizing other critical parameters. For real-time PCR multiplex applications it is recommended that a primer matrix study be performed Table b to ensure the limiting primer concentration for an endogenous control. This way the target gene amplification is not compromised by competition for reagents in the same reaction tube well . This recommendation applies to all housekeeping genes regardless of the abundance level . needed not only for rRNA but also for less abundant genes . glyceraldehyde 3-phosphate dehydrogenase cyclophilin and hypoxanthine-guanine phosphoribosyl-transferase . The range of final concentration for forward and reverse primers is 100 to 900 nM in the matrix below. Perform an initial series of experiments to find the rough range of an optimum primer concentration. Follow with a second series of experiments to fine-tune the primer concentration range. In the following example the final results suggest a forward primer concentration 304 Aoyagi .
