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稳定的 RAPD反应体系是进行 RAPD分析的关键。分别测试了模板 DNA、d NTP、Mg2 +、引物、Taq DNA聚合酶浓度和变性时间、退火温度及时间、延伸时间、循环次数等对反应结果的影响。通过实验确定了板栗最佳的 RAPD反应条件。在 5 0 μl反应体系中含有 5 0 ng模板、0 .74μmol/L随机引物、180 μmol/L d NTP、3 U Taq DNA聚合酶、2 .0 mmo1/LMg Cl2 。反应扩增程序为 :94℃预变性5 min,40次热循环 ,每个循环包括 :94℃变性 3 0 sec,3 6℃退火 45 sec,72℃延伸 90 sec,最后 72℃终延伸 7min。按照优化的 RAPD条件进行的重复实验 ,重现性良好。
A stable RAPD reaction system is the key to RAPD analysis. The effects of template DNA, dNTP, Mg2 +, primers, Taq DNA polymerase concentration and denaturation time, annealing temperature and time, extension time and number of cycles on the reaction were tested. The optimal RAPD reaction conditions of chestnut were determined experimentally. The 50 μl reaction contained 50 ng of template, 0.74 μmol / L random primer, 180 μmol / L dNTP, 3 U Taq DNA polymerase and 2.0 mmo1 / LMg Cl2. The reaction amplification procedure was as follows: pre-denaturation at 94 ° C for 5 min followed by 40 cycles of heat. Each cycle consisted of denaturation at 94 ° C for 30 sec, annealing at 36 ° C for 45 sec, extension at 72 ° C for 90 sec and final extension at 72 ° C for 7 min. Repeated experiments performed according to optimized RAPD conditions gave good reproducibility.