[医学]目的基因的获取.ppt

各种数据库查找 PCR产物的电泳 琼脂糖凝胶电泳 聚丙烯酰胺凝胶电泳 电泳缓冲液/凝胶浓度/染料/电压 上样量的选择 PCR产物量的判断 PCR产物的回收 乙醇沉淀 凝胶回收 吸附柱直接回收 * Table1. General guidelines for standard PCR primers Length: 18–30 nucleotides GC content: 40–60% Tm: Simplified formula for estimating melting temperature (Tm): Tm = 2°C x (A+T) + 4°C x (G+C) Whenever possible, design primer pairs with similar Tm values. Optimal annealing temperatures may be above or below the estimated Tm. As a starting point, use an annealing temperature 5°C below Tm. Sequence: ? Avoid complementarity of two or three bases at the 3 ends of primer pairs to reduce primer–dimer formation. ? Avoid runs of 3 or more Gs or Cs at the 3 end. ? Avoid a 3-end T. Primers with a T at the 3 end have a greater tolerance of mismatch. ? Avoid complementary sequences within a primer sequence and between the primer pair. ? Commercially available computer software can be used for primer design. Concentration: ? Spectrophotometric conversion for primers: Absorbance of 1 at 260 nm (1 A260 unit) 20–30 μg/ml ? Molar conversions: Primer length pmol/μg 20 pmol 18mer 168 119 ng 20mer 152 132 ng 25mer 121 165 ng 30mer 101 198 ng ? Use 0.1–0.5 μM of each primer in PCR. For most applications, a 0.2 μM primer concentration will be sufficient. Storage: Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/μl to avoid repeated thawing and freezing. Store all primer solutions at –20°C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen. * .Figure 1 PCR systems were amplified in the absence (Control)and presence of different impurities (concentrations indicated) under the same cycling conditions. M: markers;SDS: single-copy gene prion protein (0.75 kb); Phenol: single-copy gene BRCA1 (0.9 kb); Ethanol: interleukin 9 receptor gene (0.5 kb); NaAc: N-Ras gene (1 kb); NaCl: single-copy cystic fibrosis gene (