Molecular Biology Problem Solver 33. 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. | GC content should be between 40 and 60 . The Tm of both primers should be similar to each other and similar to the primer-binding sites at the ends of the fragment to be amplified to achieve an optimal annealing temperature and amplification. 3 -end complementarity between primers and selfcomplementarity within primers must be avoided because it may increase primer-dimer formation and reduce PCR efficiency. This is more problematic when you have a low number of target gene copies. Avoid runs of G C especially guanidine. When performing RT-PCR design primers to go across exon-exon junctions to avoid amplifying genomic DNA. Since the use of DNase has a negative effect on RNA it is better to avoid genomic DNA amplification by primer design Huang Fasco and Kaminsky 1996 . Include controls lacking RT unless you have shown that this set of primers does not amplify genomic DNA. After designing the primers search for specificity using BLAST Basic Local Alignment Search Tool a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA Appendix C . This is especially important for those genes with many pseudogenes and related genes. If you are executing RT-PCR this is essential. Even a trace amount of genomic DNA left in the RNA sample preparation can give sufficient amplification of those genes. Often these PCR products are indistinguishable by gel electrophoresis and make data interpretation difficult. You may add exogenous sequence to the 5 end of primers for cloning and other purposes. When sequence information is ambiguous substitute deoxyinosine for the unknown nucleotide and place the ambiguous sequence on the 5 end. Design and test different primers to determine which works best. Inosine is naturally found in some tRNA. It base pairs with A C and U in the translation process Martin et al. 1985 Kwok et al. 1995 . Before testing the primers with your test sample measure the quantity .
